Yeast strains autonomously producing steroids

ABSTRACT

The present invention relates to genetically modified yeast strains autonomously producing, from a simple carbon source, steroids. The invention also relates to a method for producing steroids from such yeast strains.

[0001] The present invention relates to the production of steroids inmicroorganisms, in particular yeast strains.

[0002] Steroids, in particular cholesterol-derived steroids, areinvolved in many physiological processes, among which mention may bemade of regulation of carbohydrates and cholesterol levels in thebloodstream, maintenance and development of muscle mass, and developmentof the central nervous system.

[0003] Among the drawbacks observed in the event of an imbalance ofcirculating steroid levels, mention may be made of the possibletriggering of autoimmune diseases, such as lupus, of certain cancers,for example breast cancer, of cardiovascular diseases, for exampleatherosclerosis. Problems with steroid regulation are also suspected inthe case of the triggering of certain neurological diseases, such asParkinson's disease or Alzheimer's disease.

[0004] Steroids, in particular hydrocortisone, can be used astherapeutic agents as such or as supplements in other treatments. Thus,synthetic derivatives of glucocorticoids are used for theiranti-inflammatory and, at high doses, immunosuppressive actions.

[0005] The production of steroids is partly associated with expensivemethods of extraction or synthesis. John Warcup Cornforth was the firstto carry out the complete synthesis of a steroid, cholesterol, using aenzymatic method. However, it is important to have a method forobtaining steroids of interest, in particular cholesterol derivatives,at an affordable price.

[0006] A certain number of proteins involved in steroid biosynthesishave been expressed in yeast. Thus, patent EP 360 361 demonstrates theactivity of the proteins P450 17α and P450c21 in the yeast Kluyveromyceslactis. Similarly, the possibility of in vivo conversion of11-deoxycortisol to hydrocortisone in a genetically modified yeastexpressing P450c11 have been described (Dumas et al., 1996), as has theproduction of 17α-hydroxyprogesterone from pregnenolone in yeast(Degryse et al., 1999). In addition, Duport et al., (Duport et al.,1998) describe the synthesis of pregnenolone and progesterone in agenetically modified yeast. It has also been described, in patentapplication WO 99/40203, that inactivation of the ATF2 gene in a yeaststrain makes it possible to avoid acetylation of the steroids producedby this strain.

[0007] The present invention makes it possible to carry out steroidsynthesis, by fermentation of genetically modified yeast strains, in thepresence of a simple carbon source. The method provided by the presentinvention therefore makes it possible to obtain a large amount ofsteroids of interest, at low cost, since the method uses fermentation ofyeast and the addition of a simple carbon source which is readilycommercially available.

[0008] Definitions:

[0009] According to the present invention, the expression “simple carbonsource” is intended to mean carbon sources which can be used by thoseskilled in the art for the normal growth of a yeast. It is in particularintended to denote the various assimilable sugars, such as glucose,galactose or sucrose, or molasses, or the byproducts of these sugars. Amost particularly preferred simple carbon source is ethanol andglycerol.

[0010] According to the present invention, the term “steroid derivative”is intended to denote a compound which can be obtained, in particularwith one or two enzymatic or chemical reactions, from said steroids. Itis in particular intended to denote acetylated or hydroxylated steroidsor steroids bearing a substituent such as a halogenated (fluorine,iodine) derivative, or a methyl group.

[0011] There are various types of problems to be solved in order to beable to produce steroids in a microorganism:

[0012] it is advisable to eliminate the parasitic reactions which may beobserved due to the- presence of endogenous enzymes in the microorganismchosen,

[0013] it is advisable to introduce the genes for modifying thesynthesis intermediates such that the levels of expression obtained areas close as possible to the levels observed in mammals. Thus, recreatingthe correct balances is an important conditions for the success of sucha project,

[0014] it is advisable to obtain a level of expression of the variousgenes which makes it possible to preferentially direct the biosynthesistoward the chosen steroid.

[0015] Steroid synthesis is a series of extremely complex reactionsinvolving several enzymes in order to obtain cortisol from cholesterol.

[0016] 20,22-Dihydroxycholesterol should first be produced, which isthen transformed into pregnenolone, itself hydroxylated to17α-hydroxypregnenolone. This is then transformed into17α-hydroxyprogesterone, which gives deoxycortisol, leading to cortisol(hydrocortisone).

[0017] An alternative pathway consists of the production ofpregnenolone, and then of progesterone, transformed to17α-hydroxyprogesterone.

[0018] It has been demonstrated that pregnenolone can be produced inyeast, from a simple carbon source (Duport et al., 1998, the content ofwhich is incorporated into the present application by way of reference).To do this, it was necessary to delete an endogenous pathway (bydisruption of the A22-sterol desaturase (ERG5) gene of the endogenousergosterol biosynthesis pathway), in order to obtain an accumulation ofC-22-saturated products. Specifically, the ergosterol normally producedby yeast differs from cholesterol by an unsaturation at C-7 (8) of the Bring, an unsaturation at C-22 and an additional methyl group at C-24.Thus, the products saturated at C-22 and at C-7 in the B ring can beused as substrates by the enzymes in the cortisol production chain.

[0019] The present invention makes it possible to synthesize steroids,and in particular the steroids located further downstream thanpregnenolone, simply, by fermentation of genetically modified yeaststrains, in the presence of a simple carbon source. In a particular caseof the invention, the steroids synthesized are excreted into the culturemedium, which simplifies the purification thereof. The method providedby the present invention therefore makes it possible to obtain a largeamount of steroids of interest, at low cost, since the method usesfermentation of yeasts and the addition of a simple carbon source whichis readily commercially available.

[0020] Preferably, the steroids which can be produced by the yeaststrain according to the invention are steroids included in the cortisolsynthetic pathway, as stated above. It is also possible to produce othertypes of steroids, from 17α-hydroxypregnenolone, in particulardihydroepiandrosterone (DHEA), by the action of the enzyme17α-hydroxylase and lyase, and the derived steroids (androstenediones,testosterone, etc.). These steroids can be produced by introducing theappropriate enzymes into the yeast strain, in the same way as for thestrain exemplified in the present invention.

[0021] In order to carry out the method according to the invention, theinvention also relates to a genetically modified yeast strain producinga steroid or a steroid derivative, characterized in that it allowsautonomous production from a simple carbon source.

[0022] The fact that the production is carried out autonomously meansthat there is no need to add substrates in order to obtain the steroidof interest, so that the yeast can produce it only from the startingsimple carbon source. It is also clear that the strain can produce asteroid of the metabolic pathway, using a substrate located upstream inthe metabolic pathway, insofar as the yeast strain according to thepresent invention contains all the genes required to complete themetabolic pathway for steroid production.

[0023] Preferably, the yeast strain according to the invention producesa steroid or steroid derivative which is a derivative of cholesterolmetabolism, i.e. which is part of the cholesterol metabolic chain.Cholesterol metabolism is well known to those skilled in the art, and isexplained in biochemistry and endocrinology publications.

[0024] Thus, preferably, said steroid or steroid derivative is inparticular included in the group consisting of 17α-hydroxypregnenolone,cortisol, cortisone, cortexolone, 17α-hydroxyprogesterone, andderivatives of these steroids.

[0025] It is also possible to produce pregnenolone and progesterone witha yeast strain according to the present invention.

[0026] Thus, a subject of the present invention is in particular agenetically modified yeast strain autonomously producing, from a simplecarbon source, a steroid or a steroid derivative, derived fromcholesterol metabolism, characterized in that said steroid or steroidderivative is included in the group consisting of17α-hydroxypregnenolone, hydrocortisone, cortexolone,17α-hydroxyprogesterone, and derivatives of these steroids.

[0027] As will be seen later, the yeast strain according to theinvention has at least one genetic modification chosen from a groupconsisting of disruption or inactivation of an endogenous gene,modification of the promoter of an endogenous gene, duplication of anendogenous gene, and introduction of at least one heterologous gene, inone or more copies, episomally or chromosomally.

[0028] It is, moreover, advantageous for the yeast strain of theinvention to have a combination of said gene modifications.

[0029] As explained later, in a first embodiment, the yeast strain hasat least one disruption of an endogenous gene chosen from the groupconsisting of ERG5, ATF2, GCY1, YPR1, ARE1, ARE2, ATF1 and ADE2.

[0030] In a preferred embodiment, the yeast strain according to theinvention has a disruption of the endogenous genes ERG5, ATF2, GCY1 andYPR1. As described later, these genes encode proteins which induceparasitic reactions in the yeast.

[0031] With regard to the ADE2 gene, it may also optionally bedisrupted, in particular in order to integrate a heterologous gene intothe yeast strain.

[0032] In one embodiment, the yeast strain according to the inventionhas at least one heterologous gene integrated into the chromosome, atleast one locus chosen from ADE2, HIS3, TRP1, LEU2, GCY1, ATF2 and YPR1,the integration being carried out intragenically or intergenically inthe immediate vicinity of one of the loci.

[0033] In one embodiment of the invention, said yeast strain has atleast one heterologous gene located on a multicopy plasmid or a low copyplasmid, said multicopy plasmid being chosen from yeast 2-micronreplicon-based plasmids which replicate in Saccharomyces cerevisiae andsaid low copy plasmid being chosen from plasmids based on a chromosomalARS origin of replication with a yeast centromere.

[0034] To implement an embodiment of the invention, and as developedbelow, the yeast strain according to the invention has at least oneheterologous gene or cDNA chosen from the group consisting of the geneof sterol Δ7-reductase and of the cDNAs of cytochrome P450 SCC, ofadrenodoxin, of adrenodoxin reductase, of cytochrome b5, of3β-hydrosteroid dehydrogenase isomerase, of cytochrome P450 reductase,of cytochrome P450 C17, of cytochrome P450 C21 and of cytochrome P450C11, and of the sequences encoding these proteins.

[0035] These heterologous genes or cDNAs are under the control of apromoter sequence chosen from the group consisting of the yeastendogenous promoter sequences TDH3, TEF1, PGK1, CYC1, GAL10, ATF2, TIR1,ARH1 and ADE2, and the hybrid promoter GAL10-CYC1.

[0036] It is necessary to use a terminator sequence for any heterologousgene or cDNA introduced, preferably chosen from the terminator sequencesof the endogenous genes PGK1, CYC1, ATF2, ADE2 and NCP1.

[0037] Expression cassettes or blocks are then obtained, which consistof a promoter, the heterologous gene (or cDNA, optionally encoding themature protein preceded by the ATG codon encoding methionine (Met-mat)or encoding a fusion protein optionally having signals for addressing tocellular compartments), and a terminator sequence.

[0038] In one embodiment, the yeast strain according to the inventionhas the sterol Δ7-reductase heterologous expression block integratedinto the chromosome at the ADE2 locus.

[0039] In a particular embodiment of the invention, the yeast straincomprises at least one cassette for expression of the genes encodingP450_(SCC) and adrenodoxin cofactor of P450_(SCC), located on a highcopy plasmid, and it comprises a cassette for expression of adrenodoxinreductase cofactor of P450_(SCC), located on a single copy plasmid or alow copy plasmid or integrated into the chromosome. Preferably, theseexpression cassettes contain the mature protein preceded by amethionine, and the protein is located in the cytosol.

[0040] In one embodiment, the yeast strain comprises at least oneexpression cassette chosen from the cassettes for expression of3β-hydrosteroid dehydrogenase isomerase, of cytochrome P450c17 orcytochrome P450c21, located on a high copy plasmid or a low copy plasmidor integrated into the chromosome.

[0041] In a particular embodiment, the yeast strain according to theinvention comprises at least one expression cassette for P45011β,located on a multicopy plasmid, the protein produced having a signal foraddressing to mitochondria, and/or at least one expression cassette foradrenodoxin cofactor of P45011β, located on a multicopy plasmid, with aweak promoter (i.e. the strength of which is related to that of the CYC1promoter), the protein produced having a signal for addressing tomitochondria. Preferably, the proteins are produced in the form of aprecursor, with a homologous or heterologous signal for addressing tomitochondria, the proteins taking their mature form in this cellularcompartment.

[0042] It is therefore interesting to note that, in a particularlypreferred embodiment of the invention, two copies of the gene encodingadrenodoxin are introduced into the yeast strain, one of them beingintended to express the protein in the cytosol of the cell, the otherbeing produced such that the mature protein is in the mitochondria.

[0043] In a particular embodiment, the yeast strain also comprises atleast one expression cassette (expression promoter as mentioned abovewith the coding portion of the NCP1, ATR1 and/or ATR2 gene, with its ownterminator or terminator as defined above) located on a multicopyplasmid or low copy plasmid or integrated into the chromosome. Theexpression cassettes for NCP1, ATR1 and ATR2 may in particular beintegrated at the NCP1 locus of S. cerevisiae.

[0044] In a particular embodiment, the yeast strain according to theinvention also expresses the ARH1p protein, a protein homologous tomammalian adrenodoxin reductase in yeast, at a level higher than thephysiological expression level. Overexpression of this protein can beobtained using techniques well known to those skilled in the art, forexample by introducing a new expression cassette (expression promoter,coding portion of the ARH1 gene with its own terminator or a terminatoras defined above), in addition to the endogenous gene, into the yeast.Surprisingly, it has indeed been shown that expression of the ARH1 geneat a level higher than the physiological expression level significantlyincreases the amount of steroids produced. However, this expressionshould not be too great to obtain the desired effect. Thus, if anexpression of the ARH1 protein at a level higher than a physiologicallevel is desirable, care should be taken not to overexpress this proteintoo strongly, otherwise there is a risk of losing this increase inproduction of steroids.

[0045] The yeast strain according to the present invention may bepolyploid, diploid, haploid or aneuploid in nature, without this beingharmful to the implementation of the invention.

[0046] It is preferably a strain of Saccharomyces cerevisiae, inparticular derived from one of the strains FY 1679-28c and FY 1679-18bwhich are spores of the strain FY 1679 deposited with the American TypeCulture Collection under the number 96604.

[0047] A subject of the invention is also a yeast strain, characterizedin that it is the strain CDR07 Mat-α or TGY260, deposited with the CNCMon Jan. 24, 2001, under the respective accession numbers I-2616 andI-2615. The invention also relates to a strain obtained after crossingof CDR07 Mat-α and TGY260, and optionally sporulation and transformationwith a plasmid from yeast, in particular the strains UCY2 and UCY4 andthe strains UCY3 and UCY26 described in the present invention. A subjectof the invention is also yeast strains obtained after crossing of UCY2and TGY245, and optionally sporulation and transformation with at leastone plasmid from yeast, in particular the strains UCY5, UCY6, UCY16,UCY19, UCY20, UCY24, UCY25 and UCY26, also described in the presentinvention.

[0048] It is useful for the yeast strain according to the invention tohave the elements required for excreting the steroid produced into theculture medium, in order to simplify purification of the final product.

[0049] The invention also relates to a method for producing a steroid,characterized in that it comprises the steps of fermenting a yeaststrain according to the invention in the presence of a simple carbonsource, and of recovering the steroid produced.

[0050] Finally, the subject of the invention is also a pharmaceuticalpreparation comprising a yeast strain according to the invention,optionally with a pharmaceutically acceptable excipient, such anexcipient being well known to those skilled in the art.

[0051] Although the yeast strain according to the invention produces asteroid autonomously from a simple carbon source, it is also possible toprovide it with cholesterol or a related structure, or a substratealready present as a cholesterol derivative, in order to obtain theproducts located downstream. The possibility of being able to enter atany stage, in particular at the pregnenolone level or later in themetabolic pathway of the desired steroid therefore makes it possible inparticular to be able to provide the yeast with unnatural substrates,which lead to the synthesis of unnatural and substituted steroids, inparticular fluorinated steroids.

[0052] In a first embodiment, the yeast strain according to the presentinvention in particular makes it possible to produce the desired steroid(in particular cortisol) in an amount greater than 10 mg/l, preferablygreater than 50 mg/l, more preferably 80 mg/l, more preferably 100 mg/l,and most preferably 200 mg/l.

[0053] In another embodiment, the steroid of interest (preferablyhydrocortisone) is present in a proportion greater than 20%, preferably25%, more preferably 30%, more preferably 35%, more preferably 40%, morepreferably 50%, and most preferably 65%, of the total steroids producedby the strain according to the invention (in particular the synthesisintermediates).

[0054] In order for it to be possible for the yeast strain according tothe present invention to produce the steroids of interest, it isnecessary for it to have genetic modifications. Thus, the yeast strainaccording to the invention has at least one genetic modification chosenfrom the group consisting of disruption or inactivation of an endogenousgene, modification of the promoter of an endogenous gene, duplication ofan endogenous gene, and introduction of at least one heterologous gene(in particular an expression block with homologous promoter and/orterminator and a heterologous coding portion), in one or more copies,episomally or chromosomally.

[0055] Preferably, the yeast strain according to the invention hasseveral (at least four) genetic modifications as stated above.

[0056] Thus, some endogenous genes of the yeast are favorablyinactivated or disrupted. The genes can be inactivated or disrupted byintroducing, into the coding sequence, an exogenous gene (in particularan expression block with homologous promoter and/or terminator and aheterologous coding portion) as described below, and/or a selectablemarker. It is also possible to modify the promoters of these genes inorder to decrease the level of expression.

[0057] The yeast gene ATF2 (Cauet et al., 1999) encodes an acetyltransferase which uses pregnenolone as substrate, and disruption thereofmakes it possible to eliminate this parasitic acetylation reaction, theproduct of which cannot then be used, and thus to increase the yield ofsteroid of interest. Thus, the yields can be multiplied by values ofbetween 3 and 7 after inactivation of the ATF2 gene.

[0058] The GCY1 and YPR1 genes encode aldo-keto reductases. Thesegenes—are advantageously inactivated or disrupted. These two genes arepart of a family of 6 more or less homologous genes, all six of whichare supposed to encode aldo-keto reductases. However, the products ofthese genes are the most active on the substrates envisioned herein, inparticular GCY1, and inactivation thereof is therefore extremelyadvantageous for obtaining hydrocortisone.

[0059] As specified above, it is advantageous to inactivate the ERG5gene in order to accumulate a substrate which has a structure as closeas possible to the structure of cholesterol. However, it has been shownthat the yeast according to the invention can, nevertheless, produce thesteroids of interest despite the activity of this gene. However, inorder to optimize yields, it may be useful to inactivate it by mutation,deletion and/or insertion.

[0060] It is also possible, without this being really essential for theoverall success of the steroid production with the yeast according tothe present invention, to inactivate other genes, such as ARE1, ARE2,ADE2 or ATF1. These genes all encode proteins the absence of which mayimprove the overall yield of synthesis of the steroid of interest.

[0061] As described in the article by Duport et al., cited above (Duportet al., 1998), it is indicated that the presence of an expression blockwith homologous promoter and/or terminator and a sequence encodingΔ7-reductase is useful in that it makes it possible to desaturate the7-8 double bond of ergosterol and of its derivatives, and thus to obtainone or more precursors with a structure closer to the structure ofcholesterol, the starting substrate for the production of pregnenolone.Thus, it is advantageous for the yeast strain according to the presentinvention to contain this expression block. In a particular case, saidblock for expression of Δ7-reductase is integrated into the genome ofthe yeast, preferably at the locus of the ADE2 gene, by the same tokenleading to the disruption of the ADE2 gene. The promoter used fortranscription is an inducible promoter, such as GAL10-CYC1, or aconstitutive promoter, such as the GAL10-GAL10-CYC1 promoter which isdisrupted in the strain CA10 described in Duport et al., 1998. Theprotein used is preferentially derived from Arabidopsis thaliana (butmay also be derived from a mammalian species), the cDNA being cloned inthe native form (complementary DNA just after a translation initiationmethionine), and under the control of a transcription terminator whichis conventional in yeast, such as PGK1.

[0062] It should be noted that activity of the Δ7-reductase gene inyeast has been described by Lecain et al., 1996, the technical contentof which (in particular the sequences of the Δ7-reductase gene and theconstructs and procedures) is incorporated into the present applicationby way of reference.

[0063] The first step is the production of pregnenolone, obtained afterintroducing into the yeast the enzymes for normally transformingcholesterol into pregnenolone. In the present case, this is the enzymefor cleaving the side chain (P450_(SCC) for side chain cleavage), withtwo co-enzymes (adrenodoxin, ADX, and adrenodoxin reductase, ADR). Thetransformation of the yeast with these respective expression blocks isdescribed in Duport et al., 1998, cited above.

[0064] A complementary DNA encoding the mature proteins, with amethionine added to the N-terminal end to allow translation, ispreferably used. Promoters such as the GAL10-CYC1 hybrid promoter or theTEF1 promoter are used to ensure transcription of the cDNAs.Conventional terminators, in particular the PGK1 terminator, are used.

[0065] The various cDNAs encoding the P450_(SCC), ADR or ADX proteinsmay be of vertebrate origin, for example human or bovine origin, butalso rat or fish. The genes encoding these proteins are preferablyplaced on plasmids; for P450_(SCC) and ADX, a multicopy low copy or highcopy plasmid, in particular derived from a 2 micron yeast plasmid, ispreferred, whereas a single copy plasmid or a low copy plasmid is ratherused for the expression of ADR. The expression block for ADR may also beintegrated into the chromosome of the yeast. This makes it possible tocontrol expression of the ADR, since is appears that too much expressionharms the desired scc activity.

[0066] The proteins are preferably expressed so as to be able to exerttheir activity in the cytosol.

[0067] The following step is the conversion of pregnenolone to17α-hydroxyprogesterone, by the combined action of 17α-hydroxylase(P450c17) and 3β-hydroxysteroid dehydrogenase (3β-HSD).

[0068] To express these two proteins, strong promoters, such as TEF1,TDH3 or GAL10-CYC1, are preferably used. However, a weaker promoter,such as CYC1, may also be suitable. The terminators used areconventional, and in particular come from the PGK1 or NCP1 genes. Thecomplementary DNAs encoding the complete proteins are expressed. Thespecies of origin of these proteins does not appear to modify theresults obtained, and it is thus possible to use proteins of humanorigin (in particular one or other of the two isotypes of 3β-HSD), ofbovine origin or originating from other organisms (in particular fromfish). For these two proteins, it is a question of obtaining the bestpossible expression, and they can therefore be expressed on single copyor multicopy, low copy or high copy plasmids, or by having integratedthe expression blocks into at least one chromosome of the yeast.

[0069] Conversion of the 17α-hydroxyprogesterone to deoxycortisol isthen sought, via P450c21, which allows hydroxylation at position 21. Itis a question of expressing the protein from its cDNA in the mosteffective way possible. To do this, a strong promoter (TEF1, TDH3,GAL10-CYC1, etc.), or even the CYC1 promoter, and a conventionalterminator (in particular PGK1) are used to design the transcriptionalunit. This unit is placed on a single copy or multicopy, low copy orhigh copy plasmid, or else integrated into the genome of the yeast. Itshould be noted that the species of origin appears to be important here,and that it is preferable to use P450c21 of human origin.

[0070] The deoxycortisol is then converted to cortisol, under the actionof the P450c11 system, which contains P450c11, allowing hydroxylation atthe 11-β position, and an adrenodoxin and an adrenodoxin reductase ascofactors.

[0071] The inventors of the present application have shown that theresults obtained are better when this last system is expressed in theinner membrane of yeast mitochondria. Thus, it is advantageous toproduce fusion proteins which carry, as precursor, the mitochondrialaddressing sequence of the yeast Cox6p protein precursor.

[0072] The cDNA encoding the mature proteins is also preferably used.The P450c11 protein is thus the protein of human or bovine origin or ahuman-bovine hybrid protein. The latter construct is the preferred formfor implementing the invention. The gene used in the transcriptionalunit is preferably under the control of the CYC1 promoter. It isadvantageous to place a rabbit β-globin intron and a terminator of thehuman growth hormone gene in the position 3′ of the coding sequence. Thetranscriptional unit is preferably placed on a multicopy plasmid.

[0073] The adrenodoxin is used in its mature form, placed with asequence for addressing to mitochondria, for example chosen from thoseof the percursors of the Cox6p, Cox4p, fumarase, ARH1p and F9 ATPaseproteins, and under the control of a promoter in particular chosen fromTDH3, TEF1 and CYC1. A protein of bovine or human origin is preferred. Aterminator, which may be PGK1, should be placed in the transcriptionalunit. The expression block is preferably expressed from a multicopyplasmid.

[0074] The proteins acting as an adrenodoxin reductase is the ARH1pprotein, an endogenous yeast protein, which is normally expressed in thehost's mitochondria. Preferably, the yeast is, however, transformed insuch a way that the host strain contains a natural copy of the ARH1 geneand a second copy of the ARH1 gene under the control of the CYC1promoter. The activity of this protein is essential for obtaining thedesired effect, and it has even been observed that inactivation of thegene is lethal for yeast (Lacour et al., 1998). It should be noted thatoverexpression of this gene appears to be toxic for the organism, andthat the promoter chosen should therefore allow a level of expressionwhich leads to the desired 11-β hydroxylase activity without beingdeleterious for yeast. Surprisingly, it has in fact been shown thatexpression of the ARH1 gene at a level higher than the physiologicalexpression level significantly increases the amount of steroidsproduced. However, as mentioned above, this expression should not be toogreat to obtain the desired effect. Thus, if expression of the ARH1protein at a level greater than a physiological level is desirable, careshould be taken not to overexpress this protein too strongly, otherwisethere is a risk of losing this increase in production of steroids. Byway of example, integration of the expression cassette for ARH1,comprising as promoter the CYC1 promoter, at the LEU2 locus of S.cerevisiae gives satisfactory levels of expression and results. On theother hand, integration, at the same locus, of a cassette comprising theTEF1 promoter, acknowledged to be much stronger than the CYC1 promoter,gives less advantageous results.

[0075] Thus, two copies of a transcriptional unit encoding the ADXco-enzyme protein are preferably introduced, one of them having activityoutside the mitochondria, and in particular in the cytosol, the otherhaving activity in the mitochondria of the host cell.

[0076] It is also possible to introduce other genes, in particular thegenes encoding proteins having NADPH P450 reductase activity, such asNCP1 (yeast reductase also called CPR1), ATR1 or ATR2 (plantreductases), or human reductase. These proteins improve P450c17 andP450c21 activities. Either the endogenous promoter (NCP1) is used, orthe DNAs encoding the proteins are placed under the control of promoterssuch as GAL10-CYC1, CYC1, TEF1, etc.

[0077] It is also possible to introduce the TGL1 gene, which encodes aprotein having deesterification activity, under the control of a strongpromoter such as GAL10-CYC1, on a multicopy or single copy plasmid. Thismakes it possible to reduce the effect of parasitic reactions of sterolesterification which may remain even after inactivation of ATF2, and inparticular those produced by the product of the ARE1 and ARE2 genes.

[0078] It is also possible to add a plasmid expressing cytochrome b5from yeast or another species, which is a cofactor in several of thereactions defined above.

[0079] When it is desired to produce DHEA, it is also possible tointroduce a low or high copy plasmid encoding desmolase (P450 17α),under the control of a promoter such as GAL10-CYC1, CYC1 or TEF1, with aPGK1 terminator.

[0080] It is also possible to introduce a cDNA encoding cytochromeP450c17 having lyase activity, for example that of human origin, in thepresence of an excess of NADPH P-450 reductase, for instance mammalianreductases, NCP1, ATR1 or ATR2. These transcriptional units preferablyuse a strong promoter.

[0081] Finally, it is possible to restore the activity of the endogenousERG6 and ERG2 genes, inhibited by pregnenolone, by placing them underthe control of a strong constitutive promoter.

[0082] It is also possible to introduce other heterologous genes, inparticular encoding a protein with 24,25 sterol reductase activity, orthe HMG1 gene, present in the synthetic path with cholesterol in humans.It is also advantageous to introduce the human MDR1 gene, which encodesa pump which is not inhibited by accumulation of the abnormal sterolswhich appear due to the inhibition of the ERG6 gene by pregnenolone.This makes it possible to expel these products, too great anaccumulation of which might prove to be toxic for the host cell.

[0083] It is also possible to overexpress the yeast PDR12 gene, whichwill have a detoxification effect for the steroids which may inhibit theyeast growth.

[0084] It is understood that, when reference is made to “gene” above,this is intended to mean not only the DNA fragment encoding the proteinhaving the desired activity (and especially the cDNA fragmentrepresenting in particular the mature forms), but also the promoters (inparticular TEF1, GAL10-CYC1, CYC1, TDH3) and the terminators (inparticular the PGK1). These transcriptional units are preferentiallyintroduced on low or high copy plasmids, or integrated into thechromosome of the yeast.

[0085] It is also understood that, depending on the desired aim, and inparticular the steroid that is intended to be produced, it is possibleto introduce only some of the genes encoding the various proteins ofeach step, and to provide the yeast with an intermediate in order toobtain a steroid which is downstream in the metabolic chain.

[0086] It is also easy not to introduce the genes for obtaining theproducts located downstream, and therefore to be able to stop relativelyhigh in the metabolic chain.

[0087] The yeast used to implement the present invention ispreferably:—the FY1679-28c strain, described in Duport et al., 1998,which has the genotype (MATA, rho⁻, ura3-52, trp1Δ63, leu2 Δ1, his3Δ200,GAL2, fen1). This strain has also been described by Thierry et al.,1990;

[0088] the FY1679-18b strain, having the genotype (MATa, rho⁻, ura3-52,trp1Δ63, leu2 Δ1, his3Δ200, GAL2, fen1).

[0089] These two strains therefore have an identical genotype and anopposite sign.

DESCRIPTION OF THE FIGURES

[0090]FIG. 1: Diagrammatic representation of a biosynthetic pathway forhydrocortisone as can be obtained according to the invention.

[0091]FIG. 2: Diagrammatic representation of construction of yeaststrains exemplified according to the invention.

[0092]FIG. 3: Map of the plasmid pCV29. PGK term: PGK terminator.GAL10/CYC1 prom: GAL10/CYC1 promoter. HGH term: terminator of the humangrowth hormone gene. Intron β-globin: intron of the rabbit β-globingene. CYC1 prom: CYC1 promoter. PGK term: PGK terminator. S. cerevisiae2-micron: 2-micron origin of replication of S. cerevisiae. Cox6pre:presequence of cytochrome oxidase subunit 6. Bovine/human P450 11β:bovine/human fused cDNA of P45011β. mat-ADX: mature form of ADX with anNH₂-terminal methionine. E. coli replicon: E. coli Replicon.

[0093]FIG. 4: Map of the plasmid pCC12. CYC1 prom: CYC1 promoter. PGKterm: PGK terminator. ARS CEN: S. cerevisiae chromosomal origin ofreplication. E. coli replicon: E. coli Replicon. TDH3 prom: TDH3promoter. 3β-HSDH: 3β-hydroxysteroid dehydrogenase cDNA. matADR: matureform of ADR preceded by a methionine.

[0094]FIG. 5: Map of plasmid pFM10. CYC1_(p): CYC1 promoter. P45011β:bovine/human fused cDNA of P45011β. ADE2: yeast ADE2 gene. TDH3p: TDH3promoter. 3β-HSD: 3β-hydroxysteroid dehydrogenase cDNA. R1: base forrecombination, the sequence of which is given in SEQ ID No. 39.GAL10/CYC1p: GAL10/CYC1 promoter. matADX: cDNA encoding the mature formof ADX. URA3: yeast URA3 gene. P450_(SCC): cDNA encoding the mature formof P450_(SCC) (CYP11A1). 2-micron origin: 2-micron origin of replicationof S. cerevisiae. R2: base for recombination, the sequence of which isgiven in SEQ ID No. 40.

EXAMPLES

[0095] The examples below describe an embodiment of the presentinvention and should not be considered as limiting the invention.

[0096] For the constructions, the FY1679-28c and FY1679-18b yeaststrains described above are used as starting materials.

Example 1 Disruption of the YPR1 Gene

[0097] The construct comprising interruption of the YPR1 gene (YDR368w)by the URA3 gene in the plasmid pPOLYIII was obtained by 4 successivePCR5. First, three independent PCR5 were carried out to obtain the 5′portion of the YPR1 gene (PCR 5), the functional URA3 gene bordered byYPR1 sequences (PCR 6), and the 3′ portion of the YPR1 gene (PCR 7).

[0098] The PCR5 DNA was obtained by amplification on a genomic DNAmatrix with the oligonucleotides OTG11314 (SEQ ID No. 1) and OTG11315(SEQ ID No. 2) and, similarly, the PCR7 DNA is obtained by amplificationusing the oligonucleotides OTG11316 (SEQ ID No. 3) and OTG11317 (SEQ IDNo. 4) on the same matrix.

[0099] The URA3 gene flanked by 5′ and 3′ YPR1 region is amplified usingthe oligonucleotides OTG11463 (SEQ ID No. 5) and OTG11464 (SEQ ID No. 6)on a matrix pTG10054 described in Degryse et al., 1995, incorporatedherein by way of reference as regards the description of this plasmid.

[0100] The products of PCR5, PCR6 and PCR7 were mixed in anequimolecular—fashion and then amplified by PCR using theoligonucleotides OTG11314 (SEQ ID No. 1) and OTG11317 (SEQ ID No. 4), soas to obtain a product of PCR8.

[0101] This PCR8 product was digested with the XhoI enzyme and thensubcloned into the plasmid pPOLYIII (described by Lathe et al., 1987,and incorporated herein by way of reference as regards the descriptionof this plasmid) digested with XhoI, to give the plasmid pTG12011. Theorientation of the insertion into the plasmid pPOLYIII was determined bydigestion with the NcoI and EcoRI enzymes.

[0102] The plasmid pTG12011, which allows disruption of the parasiticYPR1 gene with the URA3 gene, is digested with the XhoI enzyme. Thedigestion product is used to transform the FY1679-18b strain using thelithium chloride method well known to those skilled in the art. Thetransformants are selected on a uracil-free medium. The transformantsare analyzed by PCR amplification using the oligonucleotides which wereused to construct the plasmid pTG12011.

[0103] The clones which are positive in this test are then screened bythe 17OH-progesterone bioconversion method described below, in thepresence of glucose as carbon source. The capacity for bioconversion isanalyzed by HPLC as described by Dumas et al., 1996 and Degryse et al.,1999, the contents of which are incorporated into the presentapplication by way of reference, in particular the explanations of thebioconversion studies, or as described in Kuronen at al., 1999. A clone,TGY195#4, is selected for further characterizations. The TGY195#4 strainis transformed using both the plasmid YRp7 (Parent et al., 1985, whichis incorporated by way of reference as regards the description of thisplasmid) (1 μg) and 5 μg of plasmid pTG12045 (described below) digestedwith NotI. The transformed strains are selected on a tryptophan-freemedium. Colonies (678 colonies) are subcultured on a medium containingtrytophan (so as to eliminate the plasmid YRp7) and on a mediumcontaining tryptophan and 5-fluoroorotate (5F0) in order to select thecolonies which have lost the URA3 gene interrupting the YPR1 gene.

Example 2 Construction of Various Plasmids

[0104] For overexpression of the P450c21 protein in yeast two types ofpromoter were used, TEF1 (transcription elongation factor 1) and TDH3(glyceraldehyde-3-phosphate dehydrogenase 3). In all cases, thetranscription terminator is the PGK terminator. In these plasmids, theSalI, MluI fragment carries the human P450c21 cDNA.

[0105] a) Construction of the Plasmids pTG10470 and pTG10469

[0106] The plasmid pTG10289 was obtained by modification of pMAc21 (Wuet al., 1991) by digestion with KpnI and MluI and introduction of theoligonucleotide OTG5868 (SEQ ID No. 27).

[0107] The cDNA of this plasmid comes from the American Type CultureCollection (ATCC, Rockville, Md., USA) under the name pc21/3c. It is the1.6 Kb EcoRI-BamHI fragment which was used as a base to construct thevarious plasmids. The modifications introduced are described in thearticle above and in the article by Hu et al., 1990.

[0108] In this procedure, the noncoding portion of P450c21 of theplasmid pMAc21 which contains the expression cassette for p450c21 wasremoved, as was the KpnI site located therein.

[0109] The plasmid pTG10292 was obtained by transferring the human c21cDNA (SalI, MluI fragment) of the plasmid pTG10289 into the plasmidpTG10031 (described in Degryse et al., 1995, which is incorporated intothe application by way of reference) using the SalI and MluI sites.

[0110] The plasmid pTG10475 was obtained by PCR and recombination.Specifically, using the plasmid pTG10292, a fragment of the humanP450c21 cDNA representing approximately 250 nucleotides was amplifiedusing the oligonucleotides OTG7410 (SEQ ID No. 7) and OTG5927 (SEQ IDNo. 8). This fragment represents the coding sequence of human P450c21,of a SalI site and of the sequence AAAA.

[0111] This fragment was digested with SalI and then ligated onto thelinear fragment of pTG10292 digested with SalI, and the recombinationexperiment was then carried out in the BJ5183 strain described byDegryse et al., 1995.

[0112] The plasmid obtained, pTG10475, carries a P450c21 cDNA with acoding sequence identical to that of the natural cDNA, unlike theplasmid pMAc21, on a fragment compatible with the vectors generally usedby the inventors, i.e. a fragment bordered by the SalI and MulIrestriction sites. This fragment has the following environment aroundthe ATG codon for translation initiation:GTCGACAAAAATGCTGCTCCTGGGCCTGCTGC (SEQ ID No. 9).

[0113] Using this plasmid, the SalI, MulI fragment carrying the humanp450c21 cDNA was transferred into the plasmid pTG10158 (Degryse et al.,1995) by conventional cloning, to give the plasmid pTG10472.

[0114] This same SalI, MluI fragment of the plasmid pTG10472 was thentransferred by conventional cloning into the plasmid pTG10085 (Degryseet al., 1995), to give the plasmid pTG10469. This plasmid therefore hasthe human P450c21 cDNA under the control of the TEF1 promoter, with thePGK1 terminator.

[0115] This same fragment carrying the P450c21 cDNA on a SalI and MluIrestriction fragment is transferred into the plasmid pTG10092 byrecombination in the BJ5183 strain, to give the plasmid pTG10470(Degryse et al., 1996).

[0116] The plasmid pTG10470 therefore carries the human P450c21 cDNAunder the control of the TEF1 promoter and of a PGK1 terminator, with aURA3-d selection marker with the environment of the ATG initiator codondescribed above.

[0117] b) Construction of the Plasmid pTG12036

[0118] The plasmid pTG12036 was constructed in 4 steps from pTG10802.The plasmid pTG10801 (which is the origin of the plasmid pTG10802) is aplasmid of the pUC type into which a series of restriction sites hasbeen inserted between the XhoI and XhoI sites. This series of sitesincludes the HindIII, SnabI, ClaI and SpeI sites.

[0119] Between the HindIII and ClaI sites, the HindIII, ClaI cassette ofpTG10470, comprising the TEF1 promoter, the human P450c21 cDNA and thePGK1 terminator, was inserted between the HindIII and ClaI sites ofpTG10801, to give pTG10802.

[0120] This plasmid was then digested with XhoI and the cassetteintroduced is therefore deleted in order to introduce a PCR fragmentbordered by XhoI sites. This 2.5 Kb fragment comes from amplificationwith the pair of oligonucleotides OTG11844 (SEQ ID No. 10) and OTG11845(SEQ ID No. 11) on the plasmid pTG12010#40 (cf. below) so as to obtain afragment bordered by XhoI sites, containing the GCY1 gene interrupted bythe URA3 gene bordered in the 5′ position by a ClaI restriction site.

[0121] This fragment was cloned between the XhoI sites of the plasmidpTG10802, so as to obtain the plasmid pTG12035. The plamid pTG12010#36was used with the aim of introducing the missing HindIII site. Thisplasmid is essentially identical to pTG12010#40, but has a HindIII sitepositioned 3′ of the URA3 gene at the limit with the GCY1 gene, but doesnot have a ClaI site positioned 5′ of the URA3 gene at the junction withthe GCY1 gene (cf. below). By recombination in vivo in E. coli, betweenthe 2.2 Kb NcoI, BamH1 fragment which carries from 5′ to 3′ a fragmentof the URA3 gene, the 3′ fragment of the GCY1 gene and, finally, aportion of the plasmid pTG12035, i.e. the large 4.45 Kb StuI, AflIIfragment. The plasmid pTG12036 is obtained.

[0122] The plasmid obtained, pTG12036, has the GCY1 gene interrupted bythe URA3 gene bordered by ClaI and HindIII sites in 5′ and 3′ position,respectively. This fragment is then replaced with the expressioncassette for P450c21 carried by the 2.33 Kb ClaI, HindIII fragment ofthe plasmid pTG10469 (see above), so as to obtain the plasmid pTG12086.

[0123] c) Construction of the Plasmid pTG12045

[0124] The unique SphI site of the plasmid pPOLYIII is destroyed byinsertion of the pair of complementary oligonucleotides OTG11975 (SEQ IDNo. 12) and OTG11976 (SEQ ID No. 13).

[0125] The SphI site of pPOLYIII is destroyed and replaced with a ClaIsite, to give the plasmid pTG12040. A ClaI, EcoRI genomic DNA fragmentcorresponding to the 0.7 Kb 3′ portion of the YPR1 gene obtained byamplification with the oligonucleotides OTG11981 (SEQ ID No. 14) andOTG11982 (SEQ ID No. 15) is introduced into the plasmid pTG12040,between the unique ClaI and EcoRI sites, to give the plasmid pTG12041.

[0126] In this 2.84 Kb plasmid pTG12041, the 5′ portion of the YPR1 gene(0.66 Kb), amplified by the oligonucleotides OTG11314 (SEQ ID No. 1) andOTG11980 (SEQ ID No. 16) from wild-type yeast genomic DNA, is cloned inthe form of an XhoI, HindIII fragment, between the SalI and HindIIIsites of the plasmid pTG12041.

[0127] The 3.5 Kb plasmid pTG12042 is obtained. This plasmid carries theYPR1 gene interrupted by the ClaI and HindIII sites. The cytochromeP450c21 cassette is cloned between these sites, in the form of a 2.33 KbClaI, HindIII fragment originating from the plasmid pTG10469. Theplasmid pTG12045 is thus obtained.

[0128] d) Construction of the Plasmids pTG12010#36 and #40

[0129] The plasmid pTG 12010 was constructed based on the plasmid pUC19(Yanisch-Perron et al., 1985), whereas the plasmid pTG12011 wasconstructed based on the plasmid pPOLYIII (Lathe et al., 1987).

[0130] The construct comprising the disruption of the GCY1 gene by theURA3 gene in the plasmid pUC19 was obtained by four successive PCRamplifications. First, three independent PCR5 were carried out so as toobtain the 5′ portion of the GCY1 gene (PCR1), the functional URA3 genebordered by GCY1 sequences (PCR2), and the 3′ portion of the GCY1 gene(PCR3).

[0131] The 5′ and 3′ portions of the GCY1 gene [lacuna) using the pairsOTG11285, OTG11286 and OTG11287, OTG11289 (respectively SEQ ID No. 17 toSEQ ID No. 20) on a matrix of genomic DNA of the FY1679-28c strain.

[0132] The URA3 gene flanked by GCY1 sequences (in such a way as toobtain a deletion of part of the coding sequence of the GCY1 gene) isamplified using the oligonucleotides OTG11305 (SEQ ID No. 21) andOTG11306 (SEQ ID No. 22) from a linearized plasmid pTG10054 matrix(Degryse et al., 1995).

[0133] The buffer conditions and matrix and primer concentrationconditions for the amplification are described by the producer ormanufacturer of the Taq DNA polymerase enzyme, and in particular for theelongase enzyme developed by Life Technologies. The temperature cyclesare as follows: a first cycle of 6′30 to denature primer and matrix, andthen 30 cycles of 30 s at 93° C., 2 min at 54° C. and 3 min at 68° C.,and the final cycle is 5 min at 72° C. The PCR1, PCR2 and PCR3 productsare mixed in an equimolecular fashion and amplified again using theoligonucleotides OTG 11285 (SEQ ID No. 17) and OTG11289 (SEQ ID No. 20).The final product, PCR4, which is 2.9 Kb in size, is then subclonedbetween the KpnI and BamHI restriction sites of the plasmid pUC19, so asto obtain the plasmid pTG12010.

[0134] The structure of the plasmid was verified by restriction profileand nucleotide sequencing of the ends.

[0135] Cloning pTG12010 in fact made it possible to obtain two versionsof this plasmid, version pTG12010#40 (pTG12040 clone 40) andpTG12010#36. The initial desire was to obtain the GCY1 gene interruptedby the URA3 gene bordered by the ClaI and HindIII sites respectivelypositioned 5′ and 3′ of the gene. In fact, two different plasmids wereobtained, which differ only by the presence or absence of ClaI andHindIII sites at the ends of the URA3 gene. The plasmid pTG12010#40 hasa HindIII restriction site at the 3′ end of the URA3 gene, but no ClaIsite positioned 5′. The plasmid pTG12010#36 has no HindIII site at the3′ end, but a ClaI site at the 5′ end of the gene. This property is usedto obtain the plasmid which has the URA3 gene bordered by the HindIIIand ClaI sites, interrupting the coding sequence of GCY1.

[0136] e) Construction of the Plasmid pTG12086

[0137] This plasmid is used to integrate an expression cassette forP450c21 and also to disrupt the GCY1 gene at the same time. This plasmidwas constructed from the plasmid pTG12036 and from the plasmid pTG10614.

[0138] The construction of the plasmid pTG10614 was carried out asfollows. This plasmid was constructed from the pTG10212 (Degryse et al.,1995), which is a yeast expression plasmid based on a TDH3 promoter, aPGK1 terminator and a URA3-d selection marker.

[0139] The selection marker is replaced with the selection marker of theplasmid pTG10054 (Degryse et al., 1995) by homologous recombination inE. coli; to do this, the 2.1 Kb MluI, FspI fragment of pTG10054containing the URA3 marker flanked by recombination sequences isrecombined with the large HindIII fragment of pTG10212, to give theplasmid pTG10610 which has the same characteristics as pTG10212 (Degryseet al., 1995) with a URA3 marker in the same orientation as pTG10054.

[0140] The SalI, MulI fragment carrying the human cytochrome P450c21cDNA of the plasmid pTG10472 (cf. above) is transferred into the plasmidpTG0610, to give the plasmid pTG10614.

[0141] The ClaI, HindIII fragment of this plasmid containing, from 5′ to3′, the TDH3 promoter, the human P450c21 cDNA bordered by SalI and MluIsites, and then the PGK1 terminator is transferred into the plasmidpTG12036, to give the plasmid pTG12086 which therefore contains thesequence of the GCY1 gene interrupted by the TDH3 expression cassettefor human cytochrome P450c21.

[0142] f) Construction of the Plasmid pTG12048

[0143] The plasmid pTG12048 was constructed from the plasmids pFL26CD,pTG10925 and pTG10953.

[0144] pTG10953 is identical to the plasmid described in Lacour et al.,1998, but the TEF1 promoter which is carried by a ClaI, SalI fragment isreplaced with a CYC1 promoter (Degryse et al., 1995). The expressioncassette is carried by a NotI-NotI DNA fragment.

[0145] The plasmid pTG10925 was constructed from pFL26CD described byDuport et al., 1998. The plasmid contains a genomic fragment of yeastwhich comprises the LEU2 and NFS1 genes in 5′ to 3′ and 3′ and 5′orientation respectively. An expression cassette for ADR bordered byNotI sites was introduced into the intergenic region to give the plasmidpTG10925. This cassette (TEF1-mature bovine ADR-PGK1 terminator) wasreplaced with a new cassette comprising the ARH1 gene under the controlof the CYC1 promoter and the PGK1 terminator, to give the plasmidpTG12048. On this plasmid the LEU2 gene and the expression cassette arein the same transcriptional orientation.

Example 3 Construction of the TGY212#1 Strain

[0146] A colony is selected in the screen described in example 1. It isnamed TGY212#1. This colony is subjected to a bioconversion experimentas described previously with 100 μg/ml of 170H-progesterone substrate,and the strain is allowed to grow in a minimum medium supplemented withrequired amino acids and uracil.

[0147] This strain is capable of converting 170H-progesterone to11-deoxycortisol with an efficiency of the order of 47% over the courseof 24 hours with weak production of 4-pregnene-17α,20α-diol-3-one underthese conditions (<0.5%). Under certain conditions (defined rich mediumof the Kappeli type with galactose as carbon source and culture startedat high density: OD 600 nm=5), the capacity for reduction of the ketoneis increased to reach 11% of the starting substrate with a capacity forbioconversion reduced to 1.5% of the starting substrate.

[0148] Under these conditions, the probable presence of the GCY1 geneconstitutes a gene for the bioconversion of 170H-progesterone. It wastherefore decided to disrupt the GCY1 gene in order to prevent itsactivity.

Example 4 Construction of the TGY243 Strain

[0149] To do this, the TGY212#1 strain was transformed with 3 Hg of theplasmid pTG12010#36 linearized with the SphI and EcoRI restrictionenzymes. Twenty-seven transformants were selected on a minimum mediumsupplemented for the auxotrophies of TGY212#1, but containing no uracil.

[0150] The colonies were subjected to bioconversion tests in a minimummedium supplemented with galactose as carbon source, because the latteris a known inducer of GCY1. All the TGY243 clones exhibited a capacityto convert 170H-progesterone to 11-deoxycortisol without, however,producing detectable amounts of 4-pregnene-17α,20α-diol-3-one.

[0151] A clone, TGY243#1, was selected in order to introduce in place ofthe URA3 gene an expression cassette for human P450c21.

Example 5 Construction of the TGY245 Strain

[0152] This TGY243#1 strain is transformed with the plasmid YRp7 (1 μg)described by Parent et al., 1985 and with the plasmid pTG12086linearized with the XhoI enzyme (5 μg).

[0153] The pTG12086 transforming fragment contains the coding sequenceof GCY1 interrupted by an expression cassette for human P450c21(TDH3::human P450c21 cDNA:PGK1 terminator).

[0154] The colonies which grow in the absence of tryptophan areselected. These 381 colonies are then transferred onto a mediumcontaining tryptophan and 5-fluoroorotate. About ten colonies are thentested in a rich medium of the YPG type supplemented with tryptophan,histidine, leucine and uracil at a concentration of 50 μg/ml.

[0155] The strains are allowed to convert 170H-progesterone at aconcentration of 100 μg/ml, starting with an OD 600 nm of 0.1, for 16hours.

[0156] Among these 10 clones, one clone, TGY245#2D, is chosen based ontwo criteria: its capacity to convert 170H-progesterone to11-deoxycortisol and, secondly, the absence of formation of4-pregnene-17α,20α-diol-3-one, indicating the disruption of GCY1.

Example 6 Construction of the TGY260 Strain

[0157] The strain was constructed by transformation of TGY245 with theplasmid pTG12048 which allows overexpression of the ARH1 gene at theLEU2 locus.

[0158] The TGY260 strain was constructed from the TGY245 strain whichwas transformed with the linearized plasmid pTG12048.

[0159] This plasmid pTG12048 is an intergenic integration plasmidcarrying the LEU2 selection marker. On this plasmid, an expressioncassette for the ARH1 gene (Lacour et al., op. cit.) is integrated inthe region between the LEU2 gene and the NFS1 gene.

[0160] This cassette comprises the CYC1 promoter followed by the ARH1gene and the PGK1 terminator bordered by NotI restriction sites. Thisplasmid pTG12048 was linearized with the XhoI and SalI restrictionenzymes.

[0161] This fragment is used to transform the TGY245 strain using aconventional method of transformation with lithium chloride. Thetransformant colonies are then selected on a medium containing noleucine. A strain, TGY260, was selected.

[0162] The TGY260 strain was deposited with the CNCM [NationalCollection of Culture in Microorganisms] on Jan. 24, 2001, under thenumber I-2615.

Example 7 Construction of the CDR07matα Strain

[0163] The FY1679-28c strain was transformed with the plasmid pCD62.1,described in Duport et al., 1998 (the technical content of which forconstructing the plasmids and obtaining the strains is incorporated intothe present application by way of reference), to give the CDR01 strain.This strain is transformed with the plasmid pCD78, also described inDuport et al., and the CA03 strain is obtained.

[0164] The ERG5 gene of said CA03 strain is disrupted with a cassettewhich confers hygromycin resistance, and the CA10 strain is obtained.

[0165] This strain is crossed with an FY1679-18b parent strain, and thenallowed to sporulate. The spores are isolated according to conventionaltechniques on a minimum medium supplemented for the auxotrophies, i.e.uracil, tryptophan, leucine and histidine.

[0166] The spores are screened based on two criteria, which are nystatinresistance (12 μg/ml in minimum medium) and hygromycin resistance(100-150 μg/ml in rich medium). The spores which are resistant to thesetwo products are then analyzed by gas chromatography for the presence ofcampesterol as main membrane sterol. The presence of campesterol in themembranes of the strains, combined with an absence ofergosta-5,22-dienol, indicates that the Δ7 reductase is functioningcorrectly. A CDR07 MATα spore is selected based on the above criteria.

[0167] The CDR07 MATα strain was deposited with the CNCM on Jan. 24,2001, under the number I-2616.

Example 8 Construction of the UCY2 and UCY4 Strains

[0168] The TGY260 and CDR07 MATα strains are crossed so as to obtain theSB14 strain. This strain is then allowed to sporulate and thesegregating strains YCC4 and YCC5 are obtained (see also below).

[0169] These strains are transformed with the plasmid pLIP5 (see below)and give the YCC8 and YCC9 strains, respectively.

[0170] These strains are transformed with the plasmid pTG12093, so as toobtain the UCY2 and UCY3 strains, respectively.

[0171] The ATF2 gene is inactivated in the UCY2 strain using a plasmidand a selection with G418, and the UCY4 strain is then obtained.

Example 9 Construction of the Plasmids for Transforming the UCY2, UCY3and UCY4 Strains

[0172] a) Construction of Plasmid pCV29

[0173] The plasmids pTG10767, pTG10022 and pCD63 were used to constructpCV29 (cf. FIG. 3).

[0174] 1. Construction of the Plasmid pTG10767

[0175] The plasmid pTG10767 is an expression plasmid for P450c11 ofhuman origin. It in fact contains two expression cassettes, one forP450c11 of human origin and the other for adrenodoxin. These twoproteins are targeted to be active in the mitochondria of the yeast S.cerevisiae, by the presence of addressing sequences of the cytochromeCox6p precursor.

[0176] This expression cassette for P450c11 of human origin is borderedby two NotI restriction sites which make it possible to transport it inthe various vectors described by Degryse et al., 1995. From 5′ to 3′, itcomprises the CYC1 promoter as described in the publication above, andthen the modified human P450c11 cDNA as described above and, finally, anoncoding portion from a higher eukaryote.

[0177] The human P450c11 cDNA is that described by Chua et al., 1987,with the following modifications.

[0178] The portion encoding the signal peptide in the original cDNA wasremoved up to the BglII restriction site and replaced. This at the sametime removes 31 amino acids of the coding sequence of the mature form ofthe cDNA, to give the following NH₂-terminal protein sequence (SEQ IDNo. 23): MLSRAIFRNPVINRTLLRARPGAYHATRL TKNTFIQSRKYGTRGAAAPKAVLPEAMPRCPGNKWMRMLQIWREQGYEDLHLEVHQTFQELGPIFRYDLGGAGMVCVMLPEDVEKLQQVDSLHPHRMSLEPWVAYRQH

[0179] On the preceding line, the part in italics corresponds to theamino acid sequence of the addressing sequence of the precursor of yeastcytochrome oxidase subunit VI (COX6), the part in bold corresponds tothe NH₂-terminal portion of bovine P450c11 (Dumas et al., 1996). Thepart underlined corresponds to the beginning of the sequence identicalto that published for the human P450111 cDNA (Kawamoto et al., 1990).

[0180] The NotI cassette which carries the CYC1 promoter and thechimeric cDNA of P450111 also contains a noncoding portion located 3′ ofthe cDNA. This noncoding portion is composed of 3 elements, a noncodingportion originating from the natural cDNA and a noncoding portionoriginating from the plasmid pCMV4, the sequence of which is depositedat Genbank under the number AF239248 (Andersson et al., 1989). Inaddition, the only noncoding portions originating from pCMV4, terminatorof the human growth hormone gene and the rabbit β-globin gene intron areconserved in the final vector pCV29.

[0181] Finally, a DNA fragment carrying NotI restriction sites contains,from 5′ to 3′, the CYC1 promoter, and a chimeric cDNA framed by SalI andMluI sites containing a portion encoding the presequence of cytochromeoxidase fused to a fragment of bovine P450_(11β) up to the portioncorresponding to the restriction site, then the end of the cDNAcorresponds to the human P450_(11β1) cDNA. The noncoding portion is madeof three of mammalian origin.

[0182] This NotI cassette is transferred into the plasmid pTG10359 whichis derived from the plasmid pTG10350 (Dumas et al., 1996) digested withthe ClaI and PvuII restriction enzymes and religated on itself.

[0183] This plasmid pTG10359 also has a unique NotI restriction site.The plasmid pCV29 is a 3-cassette plasmid which contains the expressioncassettes for the mature form of ADX and the mature form of P450_(SCC)and a for of human CYP11B1 targeted to mitochrondria.

[0184] The plasmid pTG10022 is described in the publication by Degryseet al., 1995, incorporated into the present application by way ofreference, and contains a 2-micron origin of replication for the yeastS. cerevisiae, and also the elements for cloning in E. coli.

[0185] The restriction sites of this plasmid were modified with theoligonucleotides OLIP 174 and OLIP 175 (respectively SEQ ID No. 28 and29) in order to integrate therein the AscI and PmeI restriction sites.

[0186] A double expression cassette of the plasmid pCD63 described byDuport et al., 1998 was introduced at the NotI restriction site of thisplasmid. This double expression cassette, contained on a NotI fragment,contains an expression cassette in the mature form of bovine ADX and anexpression cassette in the mature form of bovine P450_(SCC) framing aURA3 selection marker. The expression cassette for cytochrome P45011βcontaining the human P45011βcDNA under the control of the CYC1 promoterand PGK1 terminator was introduced into the unique blunt-ended PmeI siteof this plasmid after filling the NotI ends with DNA polymerase Klenowfragment.

[0187] One of the plasmids obtained, pCV29, contains three expressioncassettes for ADX, P450_(SCC) (both in mature form) and also a form ofhuman P45011β targeted to mitochondria. In addition, this plasmidcarries, as a marker, the URA3 gene framed by two expression cassettes.

[0188] b) Construction of the Plasmid pCC12

[0189] The plasmid pCC12 (cf. FIG. 4) is a replicative yeast plasmidbased on the chromosomal origin of replication such as ARS1 and a CENcentromere (there are 16 of them in yeast), both from S. cerevisiae.This plasmid replicates in the form of one or two copies in yeast.

[0190] It was constructed from pFL38C2, which is derived from pFL38(Bonneaud et al., 1991), the content of which is incorporated into thepresent application by way of reference, in particular the descriptionsof the plasmids). An oligonucleotide which contains the recognitionsequence of the NotI, PacI and AscI sites (OLIP FL SEQ ID No. 24) wasintroduced into this plasmid at the HindIII site. The orientation of thepolylinker was verified by sequencing. This plasmid carries a URA3selection marker bordered by BglII sites.

[0191] The URA3 gene was replaced with a 2.7 Kb ADE2 gene obtained byPCR amplification on the genome of FY1679-28c using the oligonucleotides5′ADE2090 (SEQ ID No. 37, CGATTCGGATCCACTAGTAACGCCGTATCGTGATTAACG) and3′ADE2089 (SEQ ID No. 38, CCTCAAGGATCCTCCTGACGTAGCGCTATCCTCGGTTCTG).

[0192] To do this, the plasmid pFL38C2 was digested with the BglIIenzyme, and the URA3 BglII fragment was replaced with the 2.7 Kbfragment containing the ADE2 gene. The plasmid obtained, pAM1, serves asa basis for constructing the plasmid pCC12.

[0193] Construction of the Plasmid from the Plasmid pAM1:

[0194] The plasmid pCC12 contains two expression cassettes, one for3β-HSDH of bovine origin, the other for adrenodoxin reductase, also ofbovine origin.

[0195] The bovine 3β-HSDH cDNA was recloned by PCR from a bovine adrenalgland cDNA library. The coding sequence with the cDNA published by Zhaoet al., 1989, was not modified.

[0196] The two oligonucleotides add a SalI site and 4 adenines in frontof the ATG, to give the sequence GTCGACAAAAATG (SEQ ID No. 25). The MluIsite is located right next to the termination codon of the bovine3β-HSDH cDNA, to give the sequence: end of the cDNATGACCTGGAGTGACAATGACGCGT (SEQ ID No. 26). The sequence recognized by theMluI enzyme is ACGCGT.

[0197] The cDNA is transferred, in the form of a SalI, MluI fragment,into the plasmid pTG10212, to give the plasmid pCC4. This plasmidtherefore has a NotI fragment containing the 3β-HSDH cDNA bordered byboth a TDH3 promoter and a PGK1 terminator, positioned 5′ and 3′respectively. This expression cassette is then transferred into theplasmid pTG12018, and the cassette is thus now bordered in the 5′position by a NotI site and in the 3′ position by an AscI site. Thisfragment is then cloned into the yeast expression plasmid pAM1 in theNotI and AscI sites, to give the plasmid pCC11.

[0198] Into the NotI site of this plasmid are inserted the NotI fragmentcarrying the TEF1 promoter, the cDNA of the mature form of adrenodoxinreductase of bovine origin, and the PGK1 yeast terminator, respectivelyin this order, originating from the plasmid pTG10361.

[0199] This plasmid carries the same cDNA which is described in Dumas etal., 1996 (the content of which is incorporated into the presentapplication by way of reference, in particular the descriptions of theplasmids), except that the addressing sequence of the cytochrome oxidaseprecursor has been replaced with a methionine codon. The cDNA thereforeencodes a mature form of the adrenodoxin reductase cDNA.

Example 10 Construction of the CDR07 Strain

[0200] The CDR07 strain was obtained by crossing between the FY1679-18bMAT a strain and the CA10 MAT α strain described by Duport et al., 1998.

[0201] These two strains were first of all isolated on a rich mediumcontaining glycerol and then on a medium in a medium containingpotassium acetate as described in “Yeast Protocols Methods in Cell andMolecular Biology Edited by Ivor H Evans in 1996. Humana Press TotowaN.J. ”.

[0202] After sporulation, the tetrads were digested with zymolyase 100Tfor 30 minutes at 37° C. Several cycles of selection were applied inorder to obtain a clone which produces campesterol as the major sterol,having lost the other characteristics of CA10.

[0203] The spores are first of all selected on a minimum mediumcontaining nystatin with supplements (adenine, leucine, tryptophan,uracil, histidine). The clones resistant to nystatin and auxotrophic foradenine and leucine are then subcultured on rich medium containingadenine and hygromycin (200 μg/ml) in order to detect deletion of theERG5 gene by the hygromycin resistance gene.

[0204] Clones auxotrophic for adenine and leucine (and uracil andtryptophan) and also hygromycin-resistant and nystatin-resistant areanalyzed in terms of their sterol profile. Two clones of oppositemating-type signs, producing campesterol as the major sterol, areselected.

[0205] They are named CDR07 MAT a and CDR07 MAT α.

Example 11 Construction of the Integration Plasmids pTG12093 and pLIP5Respectively for the HIS3 and TRP1 loci

[0206] a) Construction of pLIP5

[0207] pLIP5 is a plasmid which can be used for genomic integrations atthe TRP1 locus or as a multicopy plasmid in the yeast S. cerevisiae. Thebasic plasmid which was used to construct this plasmid is the plasmidpFL45S described in Bonneaud et al., 1991 (the content of which isincorporated into the present application by way of reference, inparticular the descriptions of the plasmids).

[0208] A genomic DNA fragment corresponding to the 5′ portion upstreamof the TRP1 promoter was first cloned using the oligonucleotides OLIP21and OLIP22 (respectively SEQ ID No. 30 and 31) to carry out a PCRamplification.

[0209] The PCR product was first subcloned into pCR-Script AmpSK(+)(stratagene, La Jolla, Calif., USA). The OLIP21 and OLIP22 ends in facthave a NarI site in the 5′ position and a HindIII site in the 3′position. This NarI, HindIII fragment replaced the multiple cloningsites of the plasmid pFL45S described above.

[0210] The plasmid obtained, pLIP3, is further modified using theoligonucleotide OLIP20 (SEQ ID No. 32), which is used to replace theunique HindIII site with the unique NotI site. A fragment containing,from 5′ to 3′, the TEF1 promoter, the cytochrome P-450c17alpha cDNA andthen the PGK1 terminator, as described in Degryse et al., 1999 (thecontent of which is incorporated into the present application by way ofreference, in particular the descriptions of the plasmids), isintroduced into this NotI site.

[0211] The final plasmid pLIP5 can both be used as a multicopy plasmidbased on the TRP1 marker, when the 2-micron portion is removed, and itcan be used as a plasmid for integration at the TRP1 locus. The cassetteis thus integrated 5′ of the TRP1 gene.

[0212] b) Construction of the Plasmid pTG12093

[0213] This plasmid was a plasmid for intergenic integration at the HIS3locus. This plasmid is constructed from the plasmid pUC-HIS3 describedby Duport et al., 1998. The unique XhoI restriction site of this plasmidwas transformed into a unique NotI restriction site using a suitablelinker while at the same time destroying the XhoI site, to give theplasmid pUC19-HIS3. Into the unique NotI site of this plasmid, [lacuna]a NotI fragment originating from pTG10792. This fragment contains theTDH3 promoter and the cDNA encoding mature bovine ADX fused to theaddressing sequence of the precursor of the COX₆ cytochrome (of theyeast cytochrome oxidase subunit 6 described in Dumas et al., 1996. TheSalI, MulI restriction fragment of plasmid pTG10350 containing the cDNAdescribed previously was transferred into the plasmid pTG10211, so as toform the plasmid pTG10792.

[0214] This plasmid therefore has a 1.6 kilobase fragment containing theTDH3 promoter, the cDNA fused between mature bovine ADX and the yeastCOX₆ presequence, and the PGK1 terminator, from 5′ to 3′. This NotI-NotIfragment is inserted into the plasmid pTG12093. HIS3 and the expressioncassette are transcribed in the same orientation.

Example 12 Crossing of CDR07 MATα with TGY260 MATa

[0215] The SB14 strain (CDR07MATα×TGY260 MATa) is allowed to sporulatein a very depleted medium containing potassium acetate. The various asciare then allowed to grow in a minimum medium containing the followingproducts: uracil, histidine, tryptophan and adenine.

[0216] The spores are then selected on a correctly supplemented minimummedium containing from 8 to 12 μg/ml of nystatin. The positive sporesare then selected based on the presence of the human P450-c21 cDNA. Todo this, the clones are screened in Kappeli medium (M Arreguin deLorencez, O J Kappeli, 1987) with 2% ethanol (and 0.1% glucose) ascarbon source in the presence of 300 mg/ml of 170H-progesterone.

[0217] The bioconversion is incubated for up to 72 hours. The capacityfor bioconversion is analyzed by HPLC as described by Dumas et al., 1996and Degryse et al., 1999 (the contents of which are incorporated intothe present application by way of reference, in particular theexplanations of the bioconversion studies).

[0218] These clones are also analyzed in terms of their sterol profileby gas chromatography as described by Duport et al., 1998 (the contentof which is incorporated into the present application by way ofreference, in particular the explanations of the bioconversion studies),for the purpose of detecting campesterol and ergosta-5,22-dienol.

[0219] Three spores, YCC3, YCC4 and YCC5, are selected on the basis thatthey produce ergosta-5,22-dienol and campesterol and that they convert170H-progesterone to 11-deoxycortisol with a production efficiency of25, 120 and 42 μg/ml in 72 hours, respectively.

[0220] YCC4 and YCC5 are then selected for two further successivetransformations with the plasmids pLIP5 and pTG12093 linearized,respectively, with the ApaLl and EcoRI enzymes. The plasmids pLIP5 andpTG12093 are intergenic expression plasmids for bovine P450c17 andmitochondrial bovine ADX respectively.

[0221] pTG12093 is a plasmid for integration into the intergenic regionin the position 3′ of the HIS3 locus, whereas pLIP5 is a plasmid forintegration into the intergenic region in the position 5′ of the TRP1locus. In addition, these two plasmids carry a unique NotI site whichallows integration of an expression cassette -containing, in this order;TEF1 promoter, bovine P450c17 cDNA, PGK1 terminator for pLIP5, and inthis order; TDH3 promoter, COXVIpre::mat bovine ADX cDNA, PGK1terminator for pTG12093.

[0222] The YCC4 strain is transformed successively with the plasmidspLIP5 and pTG12093 which are linearized (with the ApaLI and EcoRIrestriction enzymes).

[0223] In the first transformation, the transformant clones are first ofall selected on a medium which does not contain tryptophan. The cloneswhich grow in the absence of tryptophan are then selected by PCR withthe oligonucleotides C17-3 and C17-5 (respectively SEQ ID No. 33 and SEQID No. 34).

[0224] A clone, YCC8, is selected for a further transformation with thelinearized plasmid pTG12093. In the same way, the clones which grow inthe absence of histidine are selected and then the presence of the ADXcDNA is verified by PCR using the oligonucleotides ADX-3 and ADX-5(respectively SEQ ID No. 35 and SEQ ID No. 36).

[0225] A clone, UCY2, is selected; its mating-type sign is MATα.

Example 13 Construction of the UCY4 Strain

[0226] The UCY2 strain has a functional ATF2 gene, that is to say mostof the pregnenolone produced is transformed to pregnenolone acetate bythe ATF2p protein which is a pregnenolone acetyl transferase, thenatural function of which in yeast is unknown (Cauet et al., 1999). Inaddition, this reaction is irreversible and therefore, once it has beenproduced, the acetyl pregnenolone can no longer be transformed intohydrocortisone. It therefore appears to be important to destroy thisactivity so as to allow more efficient production of hydrocortisone.

[0227] The ATF2 gene was therefore disrupted using a G418 resistancegene. To do this, a disruption plasmid, pAM3kanaC, was constructed,allowing introduction of a G418 resistance marker into the ATF2 gene.

[0228] This plasmid was constructed from the plasmid pAM1 (theconstruction of which was described above), and from the plasmidpTG12002, which is an expression plasmid for the ATF2 gene.

[0229] pTG12002 is an expression plasmid for ATF2 based on the plasmidpTG10260 (Degryse et al., 1995) in which the XbaI restriction site ofthe 2-micron origin of replication has been inactivated. This plasmidtherefore comprises an expression cassette for the ATF2 gene (Cauet etal., 1999) comprising the CYC1 promoter, the ATF2 gene (framed by theSalI and MluI restriction sites) and the PGK1 terminator. This cassettewas modified by PCR so as to contain the complete ATF2 gene comprisingthe promoter of the ATF2 gene, the coding sequence of the ATF2 gene, andthe terminator of the ATF2 gene, on a KpnI, NotI restriction fragment.This KpnI-NotI fragment is introduced at the KpnI-NotI sites of theplasmid pAM1, to give the plasmid pAM3.

[0230] In the plasmid pAM3, the expression cassette for G418 resistanceis introduced into the ATF2 gene, causing the inactivation thereof.

[0231] To do this, the plasmid pAM3 is digested with the AccIrestriction enzyme, and then partially with the SacI restriction enzyme.A band of approximately 7500 bp is purified on a gel. The plasmid pFA6akanMX4 (Wach et al., Methods in Microbiology Volume 26 Yeast GeneAnalysis Chapter 5 PCR-based Gene Targeting in Saccharomyces cerevisiae)is digested with SacI and AccI, and the band at 1500 bp is purified on agel. The two fragments are ligated and then transformed. A plasmid isobtained, pAM3kanaC. pAM3kanaC is digested with PvuII and NotI, and theband at 2215 bp is purified on a gel and then transformed into UCY2,which is plated out on dishes of rich medium containing 130 μg/ml ofG418. Approximately 600 clones are subcultured on this medium containingG418, and two clones are resistant to G418. A clone which does notcontain the plasmid pAM3 is conserved.

[0232] This method of gene activation is well known to those skilled inthe art.

[0233] The single clone thus obtained is allowed to carry outbioconversion with 100 μg/ml of pregnenolone in Kappeli medium.

[0234] After 24 hours, the absence of pregnenolone acetate is verifiedby extraction and gas chromatography as described in Cauet et al., 1999.This phenomenon indicates that the ATF2 gene responsible for acetylationof pregnenolone has clearly been disrupted and is no longer functional.The strain is named UCY4.

Example 14 Construction of the UCY3 and UCY26 Strains

[0235] Screening of the spores obtained by crossing the CDR07 and TGY260strains gave several strains, including the YCC4 and YCC5 strains. Asdescribed above, the YCC4 strain was transformed with a series of 2plasmids: pLIP5 and pTG12093. A spore, UCY2, was then selected (cf.example 12). In the same way, the YCC5 strain was also transformed withthe plasmids pLIP5 and pTG12093 and an additional spore, named UCY3, wasobtained. Like UCY2, UCY3 is characterized by the presence andexpression of A. thaliana Δ7 reductase measured by nystatin resistanceand the presence of brassicasterol and campesterol as major sterol ofthese recombinant yeasts. The presence of the ADX cDNA is verified byPCR using the oligonucleotides ADX-3 and ADX-5 (respectively SEQ ID No.35 and SEQ ID No. 36), and then by Western blot as described in Dumas etal., 1996, incorporated herein by way of reference as regards thedescription of this technique.

[0236] The activity of progesterone hydroxylation at position 17 and atposition 21 is verified by bioconversion of progesterone to17OH-progesterone and 11-deoxycortisol. To do this, the strain isincubated in the presence of 200 mg/l of progesterone as described inDumas et al., 1996 and Degryse et al., 1999. UCY3 is capable ofproducing 11-deoxycortisol from progesterone, indicating the presence ofthe P450c17 and P450c21 activities. The presence of4-pregnene-17α,20α-diol-3-one is also detected, indicating that at leastone of the two activities encoded by GCY1 or YPR1 is present in thestrain. To avoid accumulation of pregnenolone acetate, the pregnenoloneacetylation activity encoded by the ATF2 gene was eliminated. With thisaim, the plasmid pAM3kanaC (cf. example 13) was used to transform theUCY3 strain. pAM3kanaC is first of all digested with PvuII and NotI, theband at 2215 bp is purified on a gel and is then transformed into UCY3,which is plated on dishes of rich medium containing 130 μg/ml of G418.Colonies resistant to the G418 antibiotic, and which are no longercapable of transforming pregnenolone into pregnenolone acetate, areisolated; a colony, UCY26, is more particularly selected for furthertransformations and to test its production of hydrocortisone.

Example 15 Construction of the UCY5 Strain

[0237] With the aim of having better genetic variability, the UYC2strain (cf. example 12) was crossed with the TGY245 strain (cf. example5), and a diploid strain, YSA2-2n, was thus selected. This strain wasplaced under conditions for the production of asci and 85 asci weredissected (as described in “Yeast Protocols in Molecular Biology Volume53 p59-67, 1996”). The isolated spores are screened on the basis oftheir auxotrophic property. Thus, the clones capable of growing in theabsence of tryptophan and histidine and requiring adenine are moreparticularly selected. Expression of Δ7 reductase is verified by makingsure that the strain is resistant to nystatin and that campesterol andbrassicasterol are present in the membranes of these strains. The latteranalysis is carried out by gas chromatography of the total sterols ofthese strains as described in Duport et al., 1998. In addition, thepresence of the cDNAs encoding P450c17 and ADX is verified by PCR usingthe oligonucleotides C17-3 and C17-5 (respectively SEQ ID No. 33 and SEQID No. 34) and the oligonucleotides ADX-3 and ADX-5 (respectively SEQ IDNo. 35 and SEQ ID No. 36), respectively. Finally, the functionality ofthe GCY1 and YPR1 genes in these positive spores is verified in twoways: either by PCR or by the absence of 20-alpha-reductase activity on170H-progesterone.

[0238] Disruption of the YPR1 gene by the expression cassette for humanP450c21 in S.cerevisiae, containing the TEF1 promoter, the human P450c21cDNA and the PGK terminator, is detected by PCR using the pair ofoligonucleotides X1TEF1 and X2C21 (respectively SEQ ID No. 47 and SEQ IDNo. 48). Disruption of the GCY1 gene by the expression cassette forhuman P450c21 in S.cerevisiae, containing the TDH3 promoter, the humanP450c21 cDNA and the PGK terminator, is also detected by PCR using thepair of oligonucleotides X3TDH3 and X2C21 (respectively SEQ ID No. 49and SEQ ID No. 48).

[0239] The disappearance of the two GCY1 and YPR1 activities and thepresence of the activity corresponding to the P450c17 and P450c21activity are finally verified by bioconversion of progesterone to11-deoxycortisol under the conditions described by Dumas et al., 1996,modified by Degryse et al., 1999. The recombinant yeasts are incubatedat 30° C. in the presence of 200 mg/l of progesterone with a celldensity of 5 in a culture medium of the Kappeli type containing 2% ofgalactose or 2% of glucose as carbon source, in a volume of 10 ml. Afterincubation for 48 hours, the culture medium of these yeasts is extractedas described previously and the amount of 170H-progesterone and11-deoxycortisol produced in particular are measured by HPLC. The sporeselected no longer produces a detectable amount of4-pregnene-17α,20α-diol-3-one, but produces 17αH-progesterone and11-deoxycortisol using the two carbon sources. The clone chosen producesthe largest amount of 1-deoxycortisol.

[0240] A strain which satisfies positively all these criteria is moreparticularly selected for further transformations; this strain is namedUCY5.

Example 16 Construction of the Plasmids pFM10 and pTG10897

[0241] a) Construction of the Plasmid pFM10

[0242] The plasmid pFM10 is a vector which allows the simultaneousexpression of 4 proteins in yeast. It contains no origin of replicationfor E. coli nor an ampicillin resistance gene; this plasmid cannotreplicate in E. coli. It is obtained by recombination in yeast of twoplasmids: pFM7 and pCB12. These plasmids, unlike the plasmid pFM10, bothreplicate in E. coli since they possess the E. coli replicon. Theplasmid pFM7 also replicates in S.cerevisiae, since it also comprisesthe 2-micron origin of replication of the yeast S.cerevisiae. These twoplasmids also have the R1 and R2 sequences (respectively SEQ ID No. 39and SEQ ID No. 40) bordering the expression cassettes and also theselection markers. The R1 and R2 sequences come from the A. thalianaphotochlorophyllide oxidoreductase gene and are each approximately 300base pairs in size.

[0243] The two sequences R1 and R2 are cloned in reverse orientationinto the plasmids pFM7 and pCB12. Thus, between the R1 and R2 sequences,the pFM7 plasmid contains, in the following order: the 2-micron originof replication contained in the 2-micron fragment (fragment bordered bythe EcoRI restriction sites, as described by Urban et al., 1994), afragment bordered by NotL sites originating from the plasmid pCD63described in Duport et al., 1998 containing two expression cassettes forthe mature form of P450_(SCC) and the mature form of ADX separated bythe functional yeast URA3 gene, and then the R2 sequence. Similarly, theplasmid pCB12 contains the R1 and R2 sequences, but they are cloned inthe direction opposite to that of the plasmid pFM7. Thus, between the R2and R1 sequences, there is the expression cassette for P450_(11β), theADE2 selection marker and the expression cassette for 3β-HSD of bovineorigin. The expression cassette for P450_(11β) comes from the plasmidpCV29 and contains the CYC1 promoter, a hybrid cDNA encoding P450_(11β)and a PGK terminator. The ADE2 gene comes from the plasmid pAM1; it isthe BglII-BglII fragment. Similarly, the expression cassette for bovine3β-HSD, i.e. the TDH3 promoter, the bovine 3β-HSD cDNA and the PGKterminator, comes from the plasmid pCC12; it is the ClaI-AscI fragment.

[0244] b) Construction of the Plasmid pTG10897

[0245] The coding sequence of the ATF2 gene is amplified by PCR usingtwo 20mer oligonucleotides corresponding respectively to the 5′ and 3′portion of the coding sequence of the ATF2 gene (and also comprising thecloning sites) and using as matrix genomic DNA from the FY 1679-28cstrain. This PCR product is then digested with the ClaI and HindIIIenzymes and is cloned between the ClaI and HindIII sites of the plasmidpTG10031 (Degryse, 1995 #102], to give the plasmid pTG10885. Thisplasmid is used as a basis for the construction of a plasmid fordisrupting the ATF2 gene. The sequence of the URA3 gene was amplifiedfrom genomic DNA of the TGY156 strain (described in Cauet et al., 1999and incorporated herein by way of reference); this strain in fact has anATF2 gene interrupted by the URA3 gene. The oligonucleotides OTG10842(SEQ ID No. 41) and OTG10841 (SEQ ID No. 42) were therefore used for theamplification on a matrix of genomic DNA from the TGY156 strain, and theamplification product was used as an initiator of recombination with theplasmid pTG10885 digested with the BstXI and StuI restriction enzymes.

[0246] The plasmid pTG10897 containing the coding sequence of the ATF2gene interrupted by the URA3 gene is thus obtained. Specifically, thefunctional URA3 gene is located between 509 nucleotides of the 5′portion of the coding sequence of ATF2 and 444 nucelotides of the 3′portion of this coding sequence.

Example 17 Construction of the UCY6, UCY16, UCY16-pFM10, UCY19, UCY20,UCY24, UCY25 and UCY27 Strains

[0247] A series of new strains was constructed, based on the UCY5strain, with the aim of improving the production of steroid of interest.Specifically, the UCY5 strain does not express adrenodoxin reductase,which is an essential component of the reaction for side chain cleavageby P450_(SCC). Furthermore, as was described previously, the ATF2 geneencodes an acetyl transferase which uses pregnenolone as substrate, anddisruption thereof makes it possible to eliminate this parasiticacetylation reaction and to thus increase the yield of steroid ofinterest. Finally, the exogenous biosynthetic pathway uses theendogenous ARH1p activity, the latter being essential to yeast survival.However, this mitochondrial activity, which replaces mammalianadrenodoxin reductase, might be limiting in the strains producinghydrocortisone. A strain having certain modifications making it possibleto considerably increase the yield of production of steroid of interestwas therefore constructed based on the UCY5 strain. Thus, this strainlacks ATF2 activity, overproduces the adrenodoxin reductase protein andalso overproduces the ARH1 protein.

[0248] In order to allow ADR to be expressed in the UCY5 strain, thelatter was transformed with the vector pTG10925 (cf. example 2 f),which, when it is transformed in yeast in linear form, allowsintegration at the LEU2 locus and expression of ADR under the control ofthe TEF1 promoter. The expression of ADR was verified by Westernblotting as described in Dumas et al., 1996. A clone expressing ADR, andnamed UCY6, was more particularly used for the subsequenttransformations.

[0249] With the aim of eliminating the parasitic acetylation reactiontransforming pregnenolone into pregnenolone acetate catalyzed by theATF2 enzyme, the ATF2 gene encoding this enzyme was disrupted.Specifically, this gene was interrupted by the URA3 marker; to do this,the NotI fragment of the plasmid pTG10897, which contains the sequenceof the ATF2 gene interrupted by the functional yeast URA3 gene, wasused. This fragment is used to transform the UCY6 strain. The coloniescapable of growing in the absence of uracil are selected, and then thecapacity of these clones to transform pregnenolone into pregnenoloneacetate is measured as described by Cauet et al., 1999. A clone capableof growing in the absence of uracil and incapable of transformingpregnenolone into pregnenolone acetate is more particularly isolated.This clone is called UCY16.

[0250] Since this strain cannot be transformed with plasmids carryingonly the URA3 marker, two new plasmids were constructed: the plasmidspCB12 and pFM7. These two plasmids, when they are recombined together,make it possible to obtain the plasmid pFM10 based on the URA3 and/orADE2 markers (cf. above and FIG. 5). Thus, to obtain the plasmid pFM10,the plasmid pFM7 is linearized with the AatII restriction enzyme and thecorresponding DNA fragment is isolated on a gel. The plasmid pCB12 is,for its part, digested with BamH1 and the 9300 base pair band isisolated according to conventional molecular biology techniques.Approximately 5 μg of the pCB12 and pFM7 fragments are mixed and areused to transform the UCY16 strain. Some UCY16-pFM10 clones, capable ofgrowing in minimum medium (without uracil and amino acids), are isolatedand the corresponding strains are tested for their level of steroidproduction according to the protocol described below.

[0251] Alternatively, and with the aim of being able to subsequently usethe plasmids of the pCV29 type based on a URA3 marker, the disruption ofthe ATF2 gene in the UCY6 strain was also obtained by transforming thisstrain with the linearized plasmid pAM3kanaC (cf. example 13). TheG418-resistant colonies are then tested for the absence or presence ofpregnenolone acetyl transferase activity as described by Cauet et al.,1999. A G418-resistant colony no longer having pregnenolone acetyltransferase activity is more particularly selected. This strain iscalled UCY24.

[0252] With the aim of increasing the ARH1 activity in a straincontaining an exogenous pathway for production of hydrocortisone, theUCY5 strain was transformed with the plasmids pTG12048 or pTG12050linearized beforehand with XhoI and SapI respectively. The plasmidpTG12048 which was described above allows overexpression of the ARH1gene at the LEU2 locus under the control of the CYC1 promoter. Theplasmid pTG12050 differs from the plasmid pTG12048 only in that the CYC1promoter controlling expression of the ARH1 gene has been replaced withthe TEF1 promoter as described in Degryse et al., 1995 and Dumas et al.,1996. UCY5 was transformed with linearized pTG12048 or pTG12050. In eachof the two cases, the colonies capable of growing in the absence ofleucine were selected. In addition, the presence of the expressioncassettes for the ARH1 gene was verified by PCR. Specifically, two pairsof oligonucleotides make it possible to verify the presence of anadditional copy of the ARH1 gene (under the control of the CYC1 or TEF1promoter). First, the pair arh1D (SEQ ID No. 43) and nfs1R (SEQ ID No.44) makes it possible to verify the presence of the junction between theARH1 gene and the NFS1 gene. Secondly, the pair leu2D (SEQ ID No. 45)and arh1R (SEQ ID No. 46) makes it possible to verify the junctionbetween the LEU2 gene and the ARH1 gene.

[0253] Besides the presence of the expression cassettes for the ARH1gene encoding the ARH1 activity, the expression of the ARH1p protein wasalso tested in the clones obtained. It is not, however, easy to detectoverexpression of ARH1p. This is due to the natural presence of theARH1p protein at a low level in S. cerevisiae strains. Western blottingexperiments carried out as described in Dumas et al., 1996 make itpossible, however, to confirm the increase in the amount of ARH1p. Inaddition to the Western blotting experiments which prove to be tricky,the presence of an increased amount of ARH1p can be verified withexperiments consisting of cytochrome c reduction or of11beta-hydroxylation of 11-dioxycortisol, which experiments arereconstituted in vitro with purified mitochondria from recombinantyeasts as described in Lacour et al., 1998 and Dumas et al., 1996,respectively. Two clones, UCY19 and UCY20, respectively transformed withpTG12048 and pTG12050 and overexpressing ARH1, are more particularlyselected. The ATF2 gene was then disrupted in these strains as describedabove. Briefly, the plasmid pAM3kanaC was linearized and then used totransform the UCY19 and UCY20 strains. The clones were then selected onthe basis of their property of G418 resistance and the absence ofpregnenolone acetyl transferase activity as described by Cauet et al.,1999. Two clones derived from UCY19 and UCY20 are more particularlyselected. They are, respectively, the UCY25 and UCY27 strains (cf. FIG.2).

Example 18 Production of Steroids by the Strains According to theInvention

[0254] a) Transformation of UCY2 and UCY4 with pCV29 and pCC12

[0255] The UCY2 and UCY4 strains were transformed with the two plasmidspCV29 and pCC12, described above, the structures of which are recalledbelow and in FIGS. 3 and 4.

[0256] The plasmid pCV29 carries a 2-micron origin of replication forreplication in yeast. In addition, this plasmid carries 3 expressioncassettes respectively for the hybrid human/bovine P450_(11β) targetedto mitochondria as described above, and the mature forms (with amethionine at the NH₂-terminal end) of adrenodoxin and of P450scc. Theexpression of these three proteins is under the control of the CYC1promoter (P45011β) and the GAL10/CYC1 promoter (ADX and P450_(SCC) inmature form).

[0257] The plasmid pCC12 is a low copy plasmid which carries anexpression cassette for the mature form of bovine adrenodoxin reductaseunder the control of the TEF1 promoter and also an expression cassettefor bovine 3β-HSD under the control of the TDH3 promoter.

[0258] These strains are allowed to grow under conventional fermentationconditions in Kappeli medium with glucose as carbon source. At the endof fermentation (180 h), the steroids are extracted as describedpreviously and characterized by high pressure liquid chromatography.

[0259] The identity of the products was verified by gas chromatography,and by reverse-phase and liquid-phase high pressure chromatography,possibly coupled with mass spectrometry and nuclear magnetic resonance(cf. table 1). TABLE 1 Review of the steroids obtained (resultsexpressed in mg/l) 4-Pregnene- Pregnenolone 17α-20α- Not Strains acetateProgesterone diol-3-one identified Corticosterone HydrocortisoneUCY2/pCV29 + 18 7 5 5 12 15 pCC12 UCY4/pCV29 + 0 30 44 24 23 80 pCC12

[0260] b) Transformation of the UCY24, UCY25, UCY26 and UCY27 Strainswith pCV29 and pCC12 and Transformation of the UCY16 Strain with pFM10

[0261] These strains have many modifications and prove to be difficultto transform. Consequently, a protocol based on preparing spheroplastswas used for these transformations, as described by Burgers et al.,1987. UCY16 was transformed according to this protocol with the plasmidspFM7 and pCB12, the latter ones giving the plasmid pMF10 byrecombination in vivo in yeast. As described above, the plasmid pFM10 isa multicopy yeast plasmid which does not contain any bacterial sequencesand allows the expression of 4 heterologous proteins. These fourproteins are as seen above; a chimeric form of cytochrome P450_(11β),bovine 3β-HSD, mature ADX and mature cytochrome P450sc. Thecorresponding cDNAs are placed, respectively, under the control of theCYC1, TDH3, GAL10/CYC1 and GAL10/CYC1 promoters. The transformants areselected on a minimum medium with nothing added. The selection iscarried out only on the basis of the ADE2 marker (growth in the absenceof adenine); the presence of a functional URA3 gene in the genome, andwhich is used to inactivate the ATF2 gene, does not make it possible toselect on the basis of the URA3 gene. It is therefore necessary toscreen several colonies in order to be sure of having a plasmid pFM10which is functional in the UCY16 strain.

[0262] The UCY24, UCY25, UCY26 and UCY27 strains were transformed withthe plasmids pCV29 and pCC12 according to the same protocol forpreparing spheroplasts. As described above, the plasmid pCV29 is amulticopy plasmid for shuttling between E. coli and S.cerevisiae. Itallows the expression of cytochrome P450_(11β), mature ADX andcytochrome P450_(SCC) respectively under the control of the CYC1,GAL10/CYC1 and GAL10/CYC1 promoters (cf. FIG. 3). The pCC12 plasmid is asingle copy yeast plasmid which allows expression of mature bovine ADRand bovine 3β-HSD under the control of the TEF1 and TDH3 promoters,respectively (cf. FIG. 4).

[0263] All these strains are allowed to grow in an Erlenmeyer flask (100ml volume and containing 30 ml of culture medium) under conventionalfermentation conditions in Kappeli medium with 2% of ethanol and 0.1% ofglucose as carbon source. After culturing for 168 hours, 500 μl ofculture medium (cells+medium) are extracted twice with 4 ml ofdichloroethane. The organic phase is pooled and then separated into twoto be dried under a stream of nitrogen. The dry extract is resuspendedin 100 μl of an acetonitrile/water (50/50, volume/volume) mixture or 100μl of dichloroethane. The extracts resuspended in the acetonitrile/watermixture are processed by HPLC; this allows analysis of the compositionof various steroids (hydrocortisone, cortexolone, corticosterone,170H-progesterone, 4-pregnene-17α,20α-diol-3-one) as described in Valvoet al., 1994. The extracts resuspended in dichloroethane are analyzed bygas chromatography as described in Duport et al., 1998, the latteranalysis making it possible to measure the amount pregnenolone and ofprogesterone in the samples. For each of the strains transformed, thesteroid productivity of about ten clones is thus measured. The resultspresented below give the results of the best of the ten clones testedfor each of the UCY24, UCY25, UCY26 and UCY27 strains transformed withpCV29 and pCC12, and the UCY16 strain transformed with pFM10. TABLE 2Review of the steroids obtained (results expressed in mg/l except forthe final line: results expressed as percentage of total steroids) UCY24UCY25 UCY26 UCY27 UCY4 pCV29 + pCV29 + pCV29 + pCV29 + UCY16 pCV29 +pCC12 pCC12 pCC12 pCC12 pFM10 pCC12 17OH-Progesterone 0.6 0.6 1.0 0.60.5 0.2 Deoxycorticosterone 0.5 0.0 0.2 0.3 1 0.0 Cortexolone 4.5 2.72.1 2.0 7.6 0.7 4-Pregnene- 0.0 0.0 0.0 0.0 0.0 4.5 17α, 20α-diol-3-oneCorticosterone 7.5 10.8 7.5 9.0 8.9 3.4 Hydrocortisone 17.7 29.2 22.324.7 15.8 12.3 Total steroids 30.8 43.3 33.1 36.6 33.8 21.2 %Hydrocortisone 57 67 67 67 47 58

[0264] The UCY16 pFM10, UCY24 pCV29+pCC12, UCY25 pCV29+pCC12, UCY26pCV29+pCC12 and UCY27 pCV29+pCC12 strains presented above are thereforecapable of producing amounts of hydrocortisone greater than thoseproduced by the UCY4/pCV29+pCC12 strain. The total amount of steroidsthus increases from 21.2 μg/ml in the UCY4/pCV29+pCC12 strain to 43.3μg/ml in the UCY25 pCV29+pCC12 strain, whereas the amount ofhydrocortisone increases from 12.3 μg/ml to 29.2 μg/ml, respectively. Inaddition, in these examples, hydrocortisone represents up toapproximately 70% of the total steroids. A large increase in the amountand the quality of the hydrocortisone produced is therefore observedsince none of these strains produces the 4-pregnene-17,20-diol-3-onecontaminant. The latter, particularly advantageous characteristic is dueto the absence of the proteins encoded by the GCY1 and/or YPR1 geneswhich are responsible for the reaction of reduction of the ketone atposition 20.

[0265] It is also interesting to note that, unexpectedly, the controlledoverproduction of ARH1p under the control of the CYC1 promotersignificantly increases the amount of total steroids produced and alsothe production of hydrocortisone (cf. UCY24 pCV29+pCC12 versus UCY25pCV29+pCC12). However, this expression should not be too great to obtainthe desired effect. Thus, if expression of the ARH1 protein at a levelgreater than a physiological level is desirable, care should be takennot to overexpress this protein too strongly, otherwise there is a riskof losing this increase in production of steroids. Thus, the TEF1promoter which is acknowledged to be much stronger than CYC1 (Nacken etal., 1996) gives unsatisfactory results (cf. UCY25 pCV29+pCC12 versusUCY27 pCV29+pCC12). In conclusion, the UCY25/pCV29+pCC12 strain has anestimated potential to produce 200 mg/l of hydrocortisone in a fermenterwith a single major contaminant (corticosterone).

[0266] c) Example of Large-Volume Production of the Strains According tothe Invention

[0267] Two of the above strains were allowed to grow in a large-volumefermenter in Kappeli medium according to techniques well known to thoseskilled in the art (cf. D. Risenberg et al., 1999, for example). Theculturing technique used is fed-batch with concentrated medium. Thefermenter, having a total volume of 15 liters, contains 6 liters ofmedium at the start. The continuous addition of 4 liters of additionalconcentrated medium during the fermentation and also of pH-correctingliquids and the continuous addition of ethanol brings the final volumeto approximately 11.5 liters at the end of culturing. The UCY4pCV29+pCC12 and UCY16 pFM10 strains were thus cultured for 180 h and anincreased production in hydrocortisone could thus be obtained (cf. table3). The results which could be envisioned for the UCY24 pCV29+pCC12,UCY25 pCV29+pCC12, UCY26 pCV29+pCC12 and UCY27 pCV29+pCC12 strains wereextrapolated from these results (cf. table 3). TABLE 3 Production ofhydrocortisone by various clones obtained according to the invention(results in mg/l) Strains Hydrocortisone in mg/l UCY4 pCV29 + pCC12 80UCY16 pFM10 110  UCY24 pCV29 + pCC12 123* UCY25 pCV29 + pCC12 203* UCY26pCV29 + pCC12 155* UCY27 pCV29 + pCC12 172*

[0268] Deposition of Biological Material

[0269] The following organisms were deposited on Jan. 24, 2001, at theCollection Nationale de Cultures de Microorganismes (CNCM) [NationalCollection of Cultures and Microorganisms], 25 rue du Docteur Roux,75724 Paris Cedex 15, France, according to the provisions of the Treatyof Budapest. CDR07 MATα strain Accession number I-2616 TGY260 strainAccession number I-2615

[0270] List of the Strains Described in the Present Application

[0271] Fy1679-18b=(n)MATa ura3-52 trp1-Δ63 leu2-Δ1 his3-Δ200 fen1 GALfen^(S) ura⁻ trp⁻ leu⁻ his⁻

[0272] Fy1679-28c=(n)MATα ura3-52 trp1-Δ63 leu2-Δ1 his3-Δ200 fen1 GALfens ura⁻ trp⁻ leu⁻ his⁻

[0273] TGY195#4=Fy679-18b ypr1::URA3

[0274] TGY212-1=Fy1679-18b ypr1::TEF1_(p)::P450c21 [5x?] ura⁻ trp⁻ leu⁻his⁻

[0275] TGY243-1=TGY212-1 gcy1::URA3 trp⁻ leu⁻ his⁻

[0276] TGY245-2D=TGY243-1 gcy1::TDH3_(p)::P450c21 ura⁻ trp⁻ leu⁻ his⁻

[0277] TGY260-A=TGY245-2D LEU2::CYC1_(p)::ARH1 ura⁻ trp⁻ his⁻

[0278] CDR01=Fy1679-18b MATαLEU2::‘b-mat ADR’ ura⁻ trp⁻ his⁻

[0279] CDR07 MATα=MATαade2::GAL10/CYC1_(p)::Δ⁷ ura⁻ trp⁻ leu⁻ his⁻ ade⁻(spore of CA10×Fy1679-28c)

[0280] CDR07 MATα=MATα ade2::GAL10/CYC1_(p)::Δ⁷ ura⁻ trp⁻ leu⁻ his⁻ ade⁻(spore of CA10×Fy1679-28c)

[0281] CA03=CDR01 ade2::GAL10/CYC1_(p)::Δ⁷ ura⁻ trp⁻ ade⁻ his⁻,resistant to nystatin (Duport et al., 1998)

[0282] CA10=CA03 erg5::PGK1_(p)::hygro^(R) ura⁻ trp⁻ ade⁻ his⁻,resistant to nystatin and hygromycin (Duport et al., 1998)

[0283] SB14 (2n)=TGY260-A×CDR07 Matα

[0284] YCC4=(n) MATαade2::GAL10/CYC1_(p)::Δ⁷, LEU2::CYC1_(p)::ARH1,ypr1::TEF1_(p)::P450c21, ERG5, fen1 (?), ura⁻ trp⁻ ade⁻ his⁻′, resistantto nystatin, (spore of SB14)

[0285] YCC8=YCC4 transformed with pLIP5 (TRP1::TEF1_(p)::P450c17::PGK1_(t)), ura⁻ ade⁻ his⁻, resistant to nystatin

[0286] UCY2=YCC8. transformed with pTG12093(HIS3::TDH3_(p):CoxVI_(pre)::matADX::PGK1_(t)):HIS3::TDH3_(p)::CoxVI_(pre)::matADX::PGK1_(t),ERG5, fen1 (?), ura⁻ ade⁻, resistant to nystatin

[0287] UCY4=UCY2 atf2-Δ::G418^(R) ura⁻ ade⁻, resistant to nystatin andto G418.

[0288] YCC5=(n) MATa ade2::GAL10/CYC1_(p)::A7Reductase,LEU2::CYC1_(p)::ARH1, gcy1::TDH3_(p)::P450c21, ERG5, fen1(?), ura⁻ trp⁻ade⁻ his⁻, (resistance to nystatin, spore of SB14).

[0289] YCC9=YCC5 transformed with pLIP5(TRP1::TEF1p::P450c17::PGK1_(t)), ura⁻ ade⁻ his⁻, resistant to nystatin.

[0290] UCY3=YCC9 transformed with pTG12093(HIS3::TDH3_(p)::CoxVI_(pre)::matADX::PGKl_(t)):HIS3::TDH3_(p)::CoxVI_(pre)::matADX::PGK1_(t),ERG5, fen1 (?), ura⁻ ade, (resistant to nystatin).

[0291] UCY26⁻ UCY3 atf2-Δ::G418R ura⁻ ade⁻, (resistant to nystatin andto G418).

[0292] YSA2 (2n)=TGY245-2D×UCY2.

[0293] UCY5=(n) MATα ade2::GAL10/CYC1_(p)::Δ7Reductase,LEU2::CYC1_(p)::ARH1, ypr1::TEF1_(p)::P450c21, ERG5,gcy1::TDH3_(p)::P450c21 fen1(?), ura⁻ trp⁻ his⁻, resistant to nystatin(spore of YSA2).

[0294] UCY6=UCY5 LEU2::TEF1::matADR::PGK1_(t).

[0295] UCY16=UCY6, atf2::URA3::atf2.

[0296] UCY19=UCY5, LEU2::CYC1::ARH1.

[0297] UCY20=UCY5, LEU2:: TEF1:: ARH1.

[0298] UCY25=UCY19, atf2-Δ::G418R (resistant to nystatin and to G418).

[0299] UCY27=UCY20, atf2-Δ::G418R (resistant to nystatin and to G418).

[0300] UCY24=UCY6, atf2-Δ::G418R (resistant to nystatin and to G418).

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1. A genetically modified yeast strain autonomously producing, from a simple carbon source, a steroid or steroid derivative, derived from cholesterol metabolism, characterized in that said steroid or steroid derivative is included in the group consisting of. 17α-hydroxypregnenolone, cortisol, cortexolone, 17α-hydroxyprogesterone, and derivatives of these steroids.
 2. The yeast strain as claimed in claim 1, characterized in that it has at least one genetic modification chosen from the group consisting of disruption or inactivation of an endogenous gene, modification of the promoter of an endogenous gene, duplication of an endogenous gene, and introduction of at least one heterologous gene in an expression block, in one or more copies, episomally or chromosomally.
 3. The yeast strain as claimed in claim 2, characterized in that it has a combination of said gene modifications.
 4. The yeast strain as claimed in one of claims 1 to 3, characterized in that it has at least one disruption of an endogenous gene chosen from the group consisting of ERG5, ATF2, GCY1, YPR1, ARE1, ARE2, ATF1 and ADE2.
 5. The yeast strain as claimed in claim 4, characterized in that it has a disruption of the endogenous genes ERG5, ATF2, GCY1 and YPR1.
 6. The yeast strain as claimed in one of claims 1 to 5, characterized in that it has at least one expression block for a heterologous gene integrated into the chromosome, at least one locus chosen from ADE2, HIS3, TRP1, LEU2, GCY1, ATF2 and YPR1, the integration being carried out intragenically or intergenically in the immediate vicinity of said locus.
 7. The yeast strain as claimed in one of claims 1 to 6, characterized in that it has at least one expression block for a heterologous gene located on a multicopy plasmid or a low copy plasmid.
 8. The yeast strain as claimed in claim 7, characterized in that the multicopy plasmid is chosen from yeast 2-micron replicon-based plasmids which replicate in Saccharomyces cerevisiae and in that the low copy plasmid is chosen from plasmids based on a chromosomal ARS origin of replication with a yeast centromere.
 9. The yeast strain as claimed in one of claims 1 to 10, characterized in that it has at least one heterologous gene or cDNA in a expression block, said gene or cDNA being chosen from the group consisting of the gene of sterol Δ7-reductase, of cytochrome P450 SCC, of adrenodoxin, of adrenodoxin reductase, of 3β-hydrosteroid dehydrogenase isomerase, of cytochrome b5, of cytochrome P450 reductase, of cytochrome P450 C17, of cytochrome P450 C21 and of cytochrome P450 C11, and of the sequences encoding these proteins.
 10. The yeast strain as claimed in claim 9, characterized in that at least one heterologous gene or cDNA is under the control of a promoter sequence chosen from the group consisting of the yeast endogenous promoter sequences TDH3, TEF1, PGK1, CYC1, GAL10, ATF2, TIR1, ARH1 and ADE2, and the hybrid promoter GAL10-CYC1.
 11. The yeast strain as claimed in either of claims 9 and 10, characterized in that the terminator sequence of at least one heterologous gene or cDNA in the expression block is chosen from the terminator sequences of the endogenous genes PGK1, CYC1, ATF2, ADE2 and NCP1.
 12. The yeast strain as claimed in one of claims 1 to 11, characterized in that it has the sterol Δ7-reductase heterologous expression block integrated into the chromosome at the ADE2 locus.
 13. The yeast strain as claimed in one of claims 1 to 12, characterized in that it comprises at least one cassette for expression of the genes encoding the mature forms of P450_(SCC) and of adrenodoxin, located on a high copy plasmid.
 14. The yeast strain as claimed in one of claims 1 to 13, characterized in that it comprises a cassette for expression of adrenodoxin reductase for P450_(SCC), located on a single copy plasmid or a low copy plasmid or integrated into one or more yeast chromosomes.
 15. The yeast strain as claimed in claim 14, characterized in that said cassette for expression of adrenodoxin reductase has the elements which ensure that the protein is present in the cytosol of the host cell.
 16. The yeast strain as claimed in one of claims 1 to 15, characterized in that it comprises at least one expression cassette chosen from the cassettes for expression of 3β-hydrosteroid dehydrogenase isomerase, of cytochrome P450c17 and of cytochrome p250c21, located on a high copy plasmid.
 17. The yeast strain as claimed in one of claims 1 to 16, characterized in that it comprises at least one expression cassette for p45011β, located on a multicopy plasmid, the protein produced having a signal for addressing to mitochondria.
 18. The yeast strain as claimed in one of claims 1 to 17, characterized in that it comprises at least one expression cassette for a precursor of adrendoxin for P45011β, located on a multicopy plasmid, with a weak promoter, the protein produced having a signal for addressing to mitochondria.
 19. The yeast strain as claimed in one of claims 1 to 18, characterized in that it has at least two expression cassettes for adrenodoxin, such that one protein is active outside the mitochondria, the other being active in the mitochondria of the host cell.
 20. The yeast strain as claimed in one of claims 1 to 19, characterized in that it comprises at least one expression cassette for NCP1, ATR1 and/or ATR2, located on a single copy plasmid or integrated into the chromosome.
 21. The yeast strain as claimed in one of claims 1 to 20, characterized in that it expresses the ARH1 protein at a level higher than a physiological level.
 22. The yeast strain as claimed in claim 21, characterized in that it has, in addition to the endogenous gene, a second expression cassette for the ARH1 protein.
 23. The yeast strain as claimed in claim 22, characterized in that the expression of the ARH1 protein is placed under the control of the CYC1 promoter in said cassette.
 24. The yeast strain as claimed in one of claims 1 to 23, characterized in that it is polyploid, diploid, haploid or aneuploid.
 25. The yeast strain as claimed in one of claims 1 to 24, characterized in that it is a strain of Saccharomyces cerevisiae.
 26. The yeast strain as claimed in claim 24, characterized in that it is derived from the FY 1679-28c strain or the FY 1679-18b strain.
 27. A yeast strain, characterized in that it is the CDR07 Mat-α or TGY260 strain, deposited with the CNCM on Jan. 24, 2001, under the respective accession numbers I-2616 and I-2615, or a strain obtained after crossing of CDR07 Mat-α and TGY260, and optionally sporulation and transformation with a plasmid from yeast, in particular the UCY2 or UCY4 strain.
 28. The yeast strain as claimed in one of claims 1 to 27, characterized in that it has the elements required for excreting the steroid produced in the culture medium.
 29. A method for producing a steroid, characterized in that it comprises the steps of fermenting a yeast strain as claimed in one of claims 1 to 28 in the presence of a simple carbon source, and of recovering the steroid produced.
 30. A pharmaceutical preparation comprising a yeast strain as claimed in one of claims 1 to
 28. 